Treatment of neurological injury by administration of human umbilical cord tissue-derived cells

ABSTRACT

Methods, pharmaceutical compositions and kits for regenerating or repairing neural tissue, decreasing apoptosis and improving neurological function following injury using human umbilical cord tissue-derived cells are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation-in-part application of U.S. patentapplication Ser. No. 12/642,773, filed Dec. 19, 2009, now abandoned,which claims benefit to U.S. Provisional Patent Application No.61/139,305, filed Dec. 19, 2008, the contents of which are incorporatedby reference herein in their entirety. This application is also acontinuation-in-part application of U.S. patent application Ser. No.14/152,649, filed Jan. 1, 2014, which is a continuation of U.S.application Ser. No. 13/605,716, filed Sept. 6, 2012, now U.S. Pat. No.8,658,152, issued Feb. 25, 2014, which is a divisional of U.S.application Ser. No. 12/429,849, filed Apr. 24, 2009, now U.S. Pat. No.8,277,796, issued Oct. 2, 2012, which is a continuation of U.S.application Ser. No. 10/877,269, filed Jun. 25, 2004, now U.S. Pat. No.7,524,489, issued Apr. 28, 2009, which in turn claims benefit of U.S.Provisional Application No. 60/483,264, filed Jun. 27, 2003, the entirecontents of which are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

This invention relates to the field of cell-based or regenerativetherapy for neurological injury. In particular, the invention providespharmaceutical compositions kits and methods for the regeneration orrepair of neural tissue using cells.

BACKGROUND OF THE INVENTION

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

Neurological diseases and other disorders of the central and peripheralnervous system are among the most debilitating that can be suffered byan individual, not only because of their physical effects, but alsobecause of their permanence. In the past, a patient suffering from brainor spinal cord injury, or a neurodegenerative condition of the centralor peripheral nervous system, such as Parkinson's disease, Alzheimer'sdisease or multiple sclerosis, to name a few, held little hope forrecovery or cure.

Neurological damage and neurodegenerative diseases were long thought tobe irreversible because of the inability of neurons and other cells ofthe nervous system to grow in the adult body. However, the adultmammalian brain retains some capacity for plasticity and neuronalregeneration following injury. (See, Kolb, B, Can J Exp Psycho, 1999;53:62-76; Stroemer, R P, et al., Stroke, 1998; 29:2381-93; Walter, D H,et al., Circulation, 2002; 105(25):3017-24; Plate, K H, J NeuropatholExp Neurol, 1999; 58(4):313-20; Szpak, G M, et al., Folia Neuropathol,1999; 37(4):264-8; Jin, K, et al., Proc Natl Acad Sci USA, 2001;98(8):4710-5; Parent, J M, et al., Ann Neurol, 2002; 52(6):802-13;Stroemer, R P, et al., Stroke, 1995; 26(11):2135-44; Keyvani, K, et al.,J Neuropathol Exp Neurol, 2002; 61(10):831-40; Lois, C, et al., Science,1996; 271(5251):978-81; and, Dutton, R, et al., Dev Neurosci, 2000;22(1-2):96-105). For example, the subventricular zone (SVZ) contains apopulation of cells capable of undergoing differentiation into variouscell types, including neurons, (See, Chen, J, et al., Stroke, 2001;32:1005-1011; Evers, B M, et al., J Am Coll Surg, 2003; 197:458-478;Seyfried, D, et al., J Neurosurg, 2006; 104:313-318) and experiments ofischemic injury and traumatic brain injury (TBI) suggest that cells inthis region participate in the recovery process. Both clinical studiesand animal models suggest that there are several mechanisms involved incellular injury following intracranial hemorrhage (ICH). These include atraumatic or mechanical component, an ischemic component, and directtoxic effects of a blood clot. (See, Gong, C, et al., Neurosurgery,2001; 48:875-883; Gong, C, et al., Brain Res, 2000; 871:57-65; Hua, Y,et al., J Cereb Blood Flow Metab, 2002; 22:55-61; Matsushita, K, et al.,J Cereb Blood Flow Metab, 2000; 20:396-404; Xi, G, et al., Stroke, 2001;32:2932-2938; and, Seyfried, D, et al., J Neurosurg, 2004; 101:104-107).Clinically, ICH occurs in close proximity to the ventricular system andtherefore, recovery from injury after ICH may involve the SVZ.

Additionally, the recent advent of stem cell-based therapy for tissuerepair and regeneration provides promising treatments for a number ofneurodegenerative pathologies and other neurological disorders. Stemcells are capable of self-renewal and differentiation to generate avariety of mature neural cell lineages. Transplantation of such cellscan be utilized as a clinical tool for reconstituting a target tissue,thereby restoring physiologic and anatomic functionality. Theapplication of stem cell technology is wide-ranging, including tissueengineering, gene therapy delivery, and cell therapeutics, i.e.,delivery of biotherapeutic agents to a target location via exogenouslysupplied living cells or cellular components that produce or containthose agents.

Stem cells with neural potency have been isolated from adult tissues.For example, neural stem cells exist in the developing brain and in theadult nervous system. These cells can undergo expansion and candifferentiate into neurons, astrocytes and oligodendrocytes. However,adult neural stem cells are rare, as well as being obtainable only byinvasive procedures, and may have a more limited ability to expand inculture than do embryonic stem cells.

Other adult tissue may also yield progenitor cells useful for cell-basedneural therapy. For instance, it has been reported recently that adultstem cells derived from bone marrow and skin can be expanded in cultureand give rise to multiple lineages, including some neural lineages.

Postpartum tissues, such as the umbilical cord, have generated interestas an alternative source of stem cells. For example, methods forrecovery of stem cells by perfusion of the placenta or collection fromumbilical cord blood or tissue have been described. A limitation of stemcell procurement from these methods has been an inadequate volume ofcord blood or quantity of cells obtained, as well as heterogeneity in,or lack of characterization of, the populations of cells obtained fromthose sources.

Additionally, neuroregeneration by mesencyhmal stem cells (MSC) aftercerebral ischemia is associated with elevated levels of growth factorssuch as vascular endothelial growth factor (VEGF) and brain-derivedneurotrophic factor (BDNF) localized to the area of the injury. Inregions of the brain surrounding experimental infarction, it has beenshown that there is increased microvessel formation and evidence ofcells migrating along the microvessels, particularly cells from the SVZ.(See, Evers, B M, et al., J Am Coll Surg, 2003; 197:458-478). Also, ithas been shown that MSC are associated with increased synaptogenesis, sothat newly formed, or recovering cells exhibit more connections, whichis consistent with the observation of improved functional recovery.(See, Seyfried, D, et al., J Neurosurg, 2006; 104:313-318). The cellularrecovery process may be aided by the removal of debris and/or secretionof growth factors, thereby creating an environment inducive to neuronalcell regeneration. Given the debilitating nature of neurological injurythere is a need to develop cellular regenerative therapies to aid inrecovery.

SUMMARY OF THE INVENTION

This invention provides compositions, kits, and methods applicable tocell-based regenerative therapy for neurological injury. In particular,the invention features pharmaceutical compositions, devices and methodsfor the regeneration or repair of neural tissue using umbilical cordtissue-derived cells.

One aspect of the invention features a method of treating a patienthaving a neurological injury, the method comprising administering to thepatient umbilical cord tissue-derived cells (UTC), in an amounteffective to treat the neurodegenerative condition. In certainembodiments, the neurological injury is cerebral ischemia, reperfusionfollowing acute ischemia, perinatal hypoxic-ischemic injury, cardiacarrest, intracranial hemorrhage, intracranial lesions, whiplash orshaken infant syndrome.

Another aspect of the invention features a method of stimulatingregeneration capacity of a SVZ of a patient comprising administering tothe patient umbilical cord tissue-derived cells in an amount effectiveto increase neurogenesis, angiogenesis, or synaptogenesis in the SVZ.

Another aspect of the invention features a method of decreasingapoptosis in a damaged or injured part of a patient's brain comprisingadministering to the patient umbilical cord tissue-derived cells in anamount effective to decrease the number of apoptotic cells in thedamaged or injured part of a patient's brain.

Another aspect of the invention features a method of improvingneurological function of a patient having a neurological injurycomprising administering to the patient umbilical cord tissue-derivedcells in an amount effective to improve the neurological function.

Another aspect of the invention features a pharmaceutical compositionfor treating a patient having a neurological injury, comprising apharmaceutically acceptable carrier and umbilical cord tissue-derivedcells. The neurological injury to be treated may be cerebral ischemia,reperfusion following acute ischemia, perinatal hypoxic-ischemic injury,cardiac arrest, intracranial hemorrhage, intracranial lesions, whiplashor shaken infant syndrome.

In certain embodiments, the pharmaceutical composition comprises cellsthat have been induced in vitro to differentiate into a neural cell orother lineage prior to formulation of the composition, or cells thathave been genetically engineered to produce a gene product that promotestreatment of the neurological injury.

In certain embodiments, the pharmaceutical composition comprises atleast one other cell type, such as astrocyte, oligodendrocyte, neuron,neural progenitor, neural stem cell or other multipotent or pluripotentstem cell. In these or other embodiments, the pharmaceutical compositioncomprises at least one other agent, such as a drug for neural therapy,or another beneficial adjunctive agent such as an anti-inflammatoryagent, anti-apoptotic agents, antioxidant or growth factor.

In certain embodiments, the pharmaceutical composition is formulated foradministration by injection or infusion. Alternatively, it may comprisean implantable device in which the cells are encapsulated, or a matrixor scaffold containing the cells.

According to yet another aspect of the invention, a kit is provided fortreating a patient having a neurological injury. The kit comprises apharmaceutically acceptable carrier, a population of umbilical cordtissue-derived cells and instructions for using the kit in a method oftreating the patient. The kit may further comprise at least one reagentand instructions for culturing the umbilical cord tissue-derived cells.It may also comprise a population of at least one other cell type, or atleast one other agent for treating a neurological injury.

According to another aspect of the invention, a method is provided fortreating a patient having a neurological injury, which comprisesadministering to the patient a preparation made from umbilical cordtissue-derived cells. Such a preparation may comprise a cell lysate (orfraction thereof) of the umbilical cord tissue-derived cells, anextracellular matrix of the umbilical cord tissue-derived cells, or aconditioned medium in which the umbilical cord tissue-derived cells weregrown. In another aspect, the invention features a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apreparation made from the umbilical cord tissue-derived cells, which maybe a cell lysate (or fraction thereof) of the umbilical cordtissue-derived cells, an extracellular matrix of the umbilical cordtissue-derived cells or a conditioned medium in which the umbilical cordtissue-derived cells were grown. Kits for practicing this aspect of theinvention are also provided. These may include one or more of apharmaceutically acceptable carrier or other agent or reagent, one ormore of a cell lysate or fraction thereof, an extracellular matrix or aconditioned medium from the umbilical cord tissue-derived cells, andinstructions for use of the kit components.

In various embodiments, the umbilical cord tissue-derived cells areinduced in vitro to differentiate into a neural cell or other lineageprior to administration. In some embodiments, the cells are geneticallyengineered to produce a gene product that promotes treatment of theneurological injury, improves neurological function, and/or promotes theregeneration capacity.

In various embodiments of the invention, the umbilical cordtissue-derived cells are administered with at least one other cell type,such as an astrocyte, oligodendrocyte, neuron, neural progenitor, neuralstem cell or other multipotent or pluripotent stem cell. In theseembodiments, the other cell type can be administered simultaneouslywith, or before, or after, the umbilical cord tissue-derived cells.Likewise, in these or other embodiments, the cells are administered withat least one other agent, such as a drug for neural therapy, or anotherbeneficial adjunctive agent such as an anti-inflammatory agent,anti-apoptotic agents, antioxidant or growth factor. In theseembodiments, the other agent can be administered simultaneously with, orbefore, or after, the umbilical cord tissue-derived cells.

In some embodiments, the cells are administered at a pre-determined sitein the central or peripheral nervous system of the patient. They can beadministered by injection or infusion, or encapsulated within animplantable device, or by implantation of a matrix or scaffoldcontaining the cells.

In certain embodiments, the cells are administered at different timepoints following the neurological injury. For example, the cells may beadministered at times ranging from about 24 hours to about 168 hours(from about 1 day to about 7 days) following the injury.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the modified neurological severity score as determined bymotor, sensory, balance and reflex tests on a graded scale of 0 to 18,with 0 being a normal score and 18 being maximal deficit, at 1 day, 4days, 7 days, 14 days, 21 days and 28 days, after injury in rats treatedwith PBS or 3×10⁶ UTC at either 24 hours or 72 hours following theinjury (n=8).

FIG. 2 shows the corner test score at 1 day, 4 days, 7 days, 14 days, 21days and 28 days, after injury in rats treated with PBS or 3×10⁶ UTC ateither 24 hours or 72 hours following the injury (n=8).

FIGS. 3A-3D show BrdU incorporation in cells of the SVZ in ratsfollowing injury and subsequent treatment with PBS at 72 hours postinjury (FIG. 3A), with 3×10⁶ UTC at 72 hours post injury (FIG. 3B), withPBS at 24 hours post injury (FIG. 3C), and with 3×10⁶ UTC at 24 hourspost injury (FIG. 3D). FIG. 3E shows the mean number of BrdU positivecells in the SVZ of rats following injury and subsequent treatment witheither PBS or 3×10⁶ UTC at 72 hours post injury (n=8), and FIG. 3F showsthe mean number with either PBS or 3×10⁶ UTC at 24 hours post injury(n=8).

FIGS. 4A-D show VWF expression in blood vessels in damaged areas of therat brains following injury and subsequent treatment with PBS at 24hours post injury (FIG. 4A), with 3×10⁶ UTC at 24 hours post injury(FIG. 4B), with PBS at 72 hours post injury (FIG. 4C), and with 3×10⁶UTC at 72 hours post injury (FIG. 4D). FIG. 4E shows the mean diameter(μm) of blood vessels in damaged areas of the rat brains followinginjury and subsequent treatment with either PBS or 3×10⁶ UTC at 24 hourspost injury (n=8), and FIG. 4F shows the mean diameter (μm) with eitherPBS or 3×10⁶ UTC at 72 hours post injury (n=8).

FIG. 5A shows BrdU incorporation in endothelial cells of blood vesselsin damaged areas of the rat brains following injury. FIG. 5B shows VWFexpression in blood vessels in damaged areas of the rat brains followinginjury. FIG. 5C shows BrdU incorporation in endothelial cells that areco-localized with VWF expressing tissue in blood vessels in damagedareas of the rat brains following injury.

FIGS. 6A-D show Doublecortin (DCX) expression in the SVZ of the ratbrains following injury and subsequent treatment with PBS at 24 hourspost injury (FIG. 6A), with 3×10⁶ UTC at 24 hours post injury (FIG. 6B),with PBS at 72 hours post injury (FIG. 6C), with 3×10⁶ UTC at 72 hourspost injury (FIG. 6D). FIG. 6E shows the mean percentage of area of theSVZ of the rat brains following injury and subsequent treatment witheither PBS or 3×10⁶ UTC at 24 hours post injury (n=8) that is positivefor Doublecortin (DCX) expression, and FIG. 6F shows the mean percentageof area with either PBS or 3×10⁶ UTC at 72 hours post injury (n=8) thatis positive for Doublecortin (DCX) expression.

FIG. 7A-D show TUJ1 expression in the SVZ of the rat brains followinginjury and subsequent treatment with PBS at 24 hours post injury (FIG.7A), with 3×10⁶ UTC at 24 hours post injury (FIG. 7B), with PBS at 72hours post injury (FIG. 7C), and with 3×10⁶ UTC at 72 hours post injury(FIG. 7D). FIG. 7E shows the mean percentage of area of the SVZ of therat brains following injury and subsequent treatment with either PBS or3×10⁶ UTC at 24 hours post injury (n=8) that is positive for TUJ1expression, and FIG. 7F shows the mean percentage of area with eitherPBS or 3×10⁶ UTC at 72 hours post injury (n=8) that is positive for TUJ1expression.

FIG. 8A-D show synaptophysin expression in the boundary zone of hematomaof the rat brains following injury and subsequent treatment with PBS at24 hours post injury (FIG. 8A), with 3×10⁶ UTC at 24 hours post injury(FIG. 8B), with PBS at 72 hours post injury (FIG. 8C), and with 3×10⁶UTC at 72 hours post injury (FIG. 8D). FIG. 8E shows the mean percentageof area of the boundary zone of hematoma of the rat brains followinginjury and subsequent treatment with either PBS or 3×10⁶ UTC at 24 hourspost injury (n=8) that is positive for synaptophysin expression, andFIG. 8F shows the mean percentage of area with either PBS or 3×10⁶ UTCat 72 hours post injury (n=8) that is positive for synaptophysinexpression.

FIGS. 9A-D show TUNEL staining of apoptotic cells in the damaged area ofthe rat brains following injury and subsequent treatment with PBS at 24hours post injury (FIG. 9A), with 3×10⁶ UTC at 24 hours post injury(FIG. 9B), with PBS at 72 hours post injury (FIG. 9C), and with 3×10⁶UTC at 72 hours post injury (FIG. 9D). FIG. 9E shows the mean number ofapoptotic cells per slide in the damaged area of the rat brainsfollowing injury and subsequent treatment with either PBS or 3×10⁶ UTCat 24 hours post injury (n=8), and FIG. 9F shows the mean number witheither PBS or 3×10⁶ UTC at 72 hours post injury (n=8).

FIGS. 10A and 10B show the mean percentage of striatum lost in the ratbrains following injury and subsequent treatment with either PBS or3×10⁶ UTC at 24 hours post injury (n=8) (FIG. 10A), and with either PBSor 3×10⁶ UTC at 72 hours post injury (n=8) (FIG. 10B).

FIGS. 11A and 11B show the mNSS at 1 day, 4 days, 7 days, 14 days, 21days, 28 days, 31 days and 35 days after injury in rats treated with PBSor 3×10⁶ UTC at 7 days following the injury (n=10) (FIG. 11A), and withPBS or 3×10⁶ UTC at 3 days following the injury (n=10) (FIG. 11B).

FIGS. 12A and 12B show the corner test score at 1 day, 4 days, 7 days,14 days, 21 days, 28 days, 31 days and 35 days, after injury in ratstreated with PBS or 3×10⁶ UTC at 7 days following the injury (n=10)(FIG. 12A), and with PBS or 3×10⁶ UTC at 3 days following the injury(n=10) (FIG. 12B).

FIGS. 13A and 13B show the cylinder test score at 1 day, 4 days, 7 days,14 days, 21 days, 28 days, 31 days and 35 days, after injury in ratstreated with PBS or 3×10⁶ UTC at 7 days following the injury (n=10)(FIG. 13A), and with PBS or 3×10⁶ UTC at 3 days following the injury(n=10) (FIG. 13B).

FIGS. 14A and 14B show the adhesive test score at 1 day, 4 days, 7 days,14 days, 21 days, 28 days, 31 days and 35 days, after injury in ratstreated with PBS or 3×10⁶ UTC at 7 days following the injury (n=10)(FIG. 14A), and with PBS or 3×10⁶ UTC at 3 days following the injury(n=10) (FIG. 14B).

FIGS. 15A-15D show BrdU incorporation in cells of the SVZ in ratsfollowing injury and subsequent treatment with PBS at 72 hours postinjury (FIG. 15A), with 3×10⁶ UTC at 72 hours post injury (FIG. 15B),with PBS at 7 days post injury (FIG. 15C), and with 3×10⁶ UTC at 7 dayspost injury (FIG. 15D). FIG. 15E shows the mean number of BrdU positivecells in the SVZ of rats following injury and subsequent treatment witheither PBS or 3×10⁶ UTC at 72 hours post injury (n=8), and FIG. 15Fshows the mean number with either PBS or 3×10⁶ UTC at 7 days post injury(n=8).

FIGS. 16A-D show VWF expression in blood vessels in damaged areas of therat brains following injury and subsequent treatment with PBS at 7 dayspost injury (FIG. 16A), with 3×10⁶ UTC at 7 days post injury (FIG. 16B),with PBS at 72 hours post injury (FIG. 16C), and with 3×10⁶ UTC at 72hours post injury (FIG. 16D). FIG. 16E shows the mean diameter (μm) ofblood vessels in damaged areas of the rat brains following injury andsubsequent treatment with either PBS or 3×10⁶ UTC at 7 days post injury(n=10), and FIG. 16F shows the mean diameter (μm) with either PBS or3×10⁶ UTC at 72 hours post injury (n=10).

FIG. 17A shows BrdU incorporation in endothelial cells of blood vesselsin damaged areas of the rat brains following injury and subsequenttreatment with 3×10⁶ UTC at 3 days post injury (n=10), and FIG. 17Bshows BrdU incorporation in endothelial cells with 3×10⁶ UTC at 7 dayspost injury (n=10). FIG. 17C shows VWF expression in blood vessels indamaged areas of the rat brains following injury and subsequenttreatment with 3×10⁶ UTC at 3 days post injury (n=10), and FIG. 17Dshows VWF expression in blood vessels with 3×10⁶ UTC at 3 days postinjury (n=10). FIG. 17E shows BrdU incorporation in endothelial cellsthat are co-localized with VWF expressing tissue in blood vessels indamaged areas of the rat brains following injury and subsequenttreatment with 3×10⁶ UTC at 3 days post injury (n=10), and FIG. 17Fshows BrdU incorporation in endothelial cells with 3×10⁶ UTC at 7 dayspost injury (n=10).

FIGS. 18A-D show TUJ1 expression in the SVZ of the rat brains followinginjury and subsequent treatment with PBS at 7 days post injury (FIG.18A), with 3×10⁶ UTC at 7 days post injury (FIG. 18B), with PBS at 3days post injury (FIG. 18C), with 3×10⁶ UTC at 3 days post injury (FIG.18D). FIG. 18E shows the mean percentage of area of the SVZ of the ratbrains following injury and subsequent treatment with either PBS or3×10⁶ UTC at 7 days post injury (n=10) that is positive for TUJ1expression, and FIG. 18F shows the mean percentage of area with eitherPBS or 3×10⁶ UTC at 72 hours post injury (n=10) that is positive forTUJ1 expression.

FIGS. 19A-D show synaptophysin expression in the boundary zone ofhematoma of the rat brains following injury and subsequent treatmentwith PBS at 72 hours post injury (FIG. 19A), with 3×10⁶ UTC at 72 hourspost injury (FIG. 19B), with PBS at 7 days post injury (FIG. 19C), with3×10⁶ UTC at 7 days post injury (FIG. 19D). FIG. 19E shows the meanpercentage of area of the boundary zone of hematoma of the rat brainsfollowing injury and subsequent treatment with either PBS or 3×10⁶ UTCat 72 hours post injury (n=10) that is positive for synaptophysinexpression, and FIG. 19F shows the mean percentage of area with eitherPBS or 3×10⁶ UTC at 7 days post injury (n=10) that is positive forsynaptophysin expression.

FIGS. 20A-D show TUNEL staining of apoptotic cells in the damaged areaof the rat brains following injury and subsequent treatment with PBS at7 days post injury (FIG. 20A), with 3×10⁶ UTC at 7 days post injury(FIG. 20B), with PBS at 72 hours post injury (FIG. 20C), with 3×10⁶ UTCat 72 hours post injury (FIG. 20D). FIG. 20E shows the mean number ofapoptotic cells per slide in the damaged area of the rat brainsfollowing injury and subsequent treatment with either PBS or 3×10⁶ UTCat 7 days post injury (n=10), and FIG. 20F shows the mean number witheither PBS or 3×10⁶ UTC at 72 hours post injury (n=10).

FIG. 21A shows the mean percentage of striatum lost in the rat brainsfollowing injury and subsequent treatment with either PBS or 3×10⁶ UTCat 7 days post injury (n=10), and FIG. 21B shows the mean percentagewith either PBS or 3×10⁶ UTC at 72 hours post injury (n=10).

FIG. 22 shows the modified neurological severity score at 1 day, 4 days,7 days, 14 days, 21 days, 28 days, and 35 days after injury in ratstreated with PBS or 4×10⁶ UTC or MSC at 24 hours following the injury(n=8).

FIG. 23 shows the Morris Water Maze score at 31 days, 32 days, 33 days,34 days and 35 days after injury in rats treated with PBS or 4×10⁶ UTCor MSC at 24 hours following the injury (n=8).

FIG. 24 shows the lesion volume as percent of brain hemisphere afterinjury in rats treated with PBS or 4×10⁶ UTC or MSC at 24 hoursfollowing the injury (n=8).

FIG. 25A shows the E5204 antibody staining to identify UTC at 35 daysafter injury in rats treated with PBS or 4×10⁶ UTC or MSC at 24 hoursfollowing the injury (n=8). No positively stained cells were found withthe PBS control. FIG. 25B shows the E5204 antibody staining to identifyMSC at 35 days after injury in rats treated with PBS or 4×10⁶ UTC or MSCat 24 hours following the injury (n=8).

FIG. 26A shows BrdU positive UTC cells 35 days after injury in ratstreated with PBS or 4×10⁶ UTC or MSC at 24 hours following the injury.FIG. 26B shows BrdU positive MSC cells 35 days after injury in ratstreated with PBS or 4×10⁶ UTC or MSC at 24 hours following the injury.

FIG. 27 shows the number of BrdU positive cells per mm² in the lesionboundary zone 35 days after injury in rats treated with PBS or 4×10⁶ UTCor MSC at 24 hours following the injury (n=8).

FIG. 28 shows the number of BrdU positive cells per mm² in the DentateGyrus 35 days after injury in rats treated with PBS or 4×10⁶ UTC or MSCat 24 hours following the injury (n=8).

FIG. 29A shows vWF stained vessels 35 days after injury in rats treatedwith 4×10⁶ UTC at 24 hours following the injury. FIG. 29B shows vWFstained vessels 35 days after injury in rats treated with 4×10⁶ MSC at24 hours following the injury.

FIG. 30 shows the number of vWF positive vessels in the lesion boundaryzone 35 days after injury in rats treated with PBS or 4×10⁶ UTC or MSCat 24 hours following the injury (n=8).

FIG. 31 shows the number of vWF positive vessels in the Dentate Gyrus 35days after injury in rats treated with PBS or 4×10⁶ UTC or MSC at 24hours following the injury (n=8).

FIG. 32A shows BrdU and Map-2 positive double stained cells andBrdU-only positive stained cells in the lesion boundary zone. FIG. 32Bshows BrdU and Map-2 positive double stained cells and BrdU-onlypositive stained cells in the dentate gyms.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description of the illustrative embodiments,reference is made to the accompanying drawings that form a part hereof.These embodiments are described in sufficient detail to enable thoseskilled in the art to practice the invention, and it is understood thatother embodiments may be utilized and that logical structural,mechanical, electrical, and chemical changes may be made withoutdeparting from the spirit or scope of the invention. To avoid detail notnecessary to enable those skilled in the art to practice the embodimentsdescribed herein, the description may omit certain information known tothose skilled in the art. The following detailed description is,therefore, not to be taken in a limiting sense.

Various terms used throughout the specification and claims are definedas set forth below.

The terms “individual,” “patient” or “subject” as used herein generallyrefer to any form of animal, including mammals, such as humans andmonkeys, who are treated with the pharmaceutical or therapeuticcompositions or in accordance with the methods described.

“Stem cells” are undifferentiated cells defined by the ability of asingle cell both to self-renew, and to differentiate to produce progenycells, including self-renewing progenitors, non-renewing progenitors,and terminally differentiated cells. Stem cells are also characterizedby their ability to differentiate in vitro into functional cells ofvarious cell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. “Totipotent” cells are able to give rise to all embryonicand extraembryonic cell types. “Pluripotent” cells are able to give riseto all embryonic cell types. “Multipotent” cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare “oligopotent” can give rise to a more restricted subset of celllineages than multipotent stem cells, and cells that are “unipotent” areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An “adult stem cell” is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An “embryonic stem cell” is a pluripotent cell from the innercell mass of a blastocyst-stage embryo. A “fetal” stem cell is one thatoriginates from fetal tissues or membranes. A “postpartum stem cell” isa multipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the umbilical cordand the placenta. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Various terms are used to describe cells in culture. “Cell culture”refers generally to cells taken from a living organism and grown undercontrolled conditions (“in culture” or “cultured”). A “primary cellculture” is a culture of cells, tissues, or organs taken directly froman organism(s) before the first subculture. Cells are “expanded” inculture when they are placed in a growth medium under conditions thatfacilitate cell growth and/or division, resulting in a larger populationof the cells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as “doubling time”.

The term “mesenchymal stem cells” (MSCs) refers to cells from theimmature embryonic connective tissue. A number of cell types come frommesenchymal stem cells, including chondrocytes, which produce cartilage.

The term “subventricular zone” (SVZ) refers to a paired brain structuresituated throughout the lateral walls of the lateral ventricles. Thelateral ventricles are part of the ventricular system of the brain.Classified as part of the telencephalon, they are the largest of theventricles. The lateral ventricles connect to the central thirdventricle through the interventricular foramina of Monro. Along with thesubgranular zone of dentate gyms, the subventricular zone serves as asource of neural stem cells in the process of adult neurogenesis.

The term “standard growth conditions” as used herein refers to culturingof cells at 37° C., in a standard atmosphere comprising 5% CO₂ andrelative humidity maintained at about 100%. While the foregoingconditions are useful for culturing, it is to be understood that suchconditions are capable of being varied by the skilled artisan who willappreciate the options available in the art for culturing cells.

The cells used in the present invention are generally referred to as“postpartum cells” or “postpartum-derived cells” (PPDC). The cells aremore specifically “umbilicus-derived cells” or “umbilical cord-derivedcells” (UDC), or “umbilical cord tissue-derived cells” (UTC). Inaddition, the cells may be described as being stem or progenitor cells,the latter term being used in the broad sense. The term “derived” isused to indicate that the cells have been obtained from their biologicalsource and grown or otherwise manipulated in vitro (e.g., cultured in agrowth medium to expand the population and/or to produce a cell line).The in vitro manipulations of umbilical stem cells and the uniquefeatures of the umbilicus-derived cells of the present invention aredescribed in detail below.

“Differentiation” is the process by which an unspecialized(“uncommitted”) or less specialized cell acquires the features of aspecialized cell, such as a nerve cell or a muscle cell, for example. A“differentiated” cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed,” when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. “De-differentiation” refers to theprocess by which a cell reverts to a less specialized (or committed)position within the lineage of a cell. As used herein, the “lineage” ofa cell defines the heredity of the cell, i.e., which cells it came fromand what cells it can give rise to. The lineage of a cell places thecell within a hereditary scheme of development and differentiation.

In a broad sense, a “progenitor cell” is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a “non-renewing progenitor cell” or as an“intermediate progenitor or precursor cell”.

As used herein, the phrase “differentiates into a neural lineage orphenotype” refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia.

The term “cell line” generally refers to a population of cells formed byone or more subcultivations of a primary cell culture. Each round ofsubculturing is referred to as a passage. When cells are subcultured,they are referred to as having been “passaged”. A specific population ofcells, or a cell line, is sometimes referred to or characterized by thenumber of times it has been passaged. For example, a cultured cellpopulation that has been passaged ten times may be referred to as a P10culture. The primary culture, i.e., the first culture following theisolation of cells from tissue, is designated P0. Following the firstsubculture, the cells are described as a secondary culture (P1 orpassage 1). After the second subculture, the cells become a tertiaryculture (P2 or passage 2), and so on. It will be understood by those ofskill in the art that there may be many population doublings during theperiod of passaging; therefore, the number of population doublings of aculture is greater than the passage number. The expansion of cells(i.e., the number of population doublings) during the period betweenpassaging depends on many factors, including, but not limited to, theseeding density, substrate, medium, growth conditions, and time betweenpassaging.

As used herein, the term “growth medium” generally refers to a mediumsufficient for the culturing of umbilical cord tissue-derived cells. Inparticular, one medium for the culturing of the cells of the inventioncomprises Dulbecco's Modified Essential Media (DMEM). Particularlypreferred is DMEM-low glucose (DMEM-LG) (Invitrogen, Carlsbad, Calif.).The DMEM-LG is preferably supplemented with serum, most preferably fetalbovine serum or human serum. Typically, 15% (v/v) fetal bovine serum(e.g. defined fetal bovine serum, Hyclone, Logan Utah) is added, alongwith antibiotics/antimycotics (preferably 100 Unit/milliliterpenicillin, 100 milligrams/milliliter streptomycin, and 0.25microgram/milliliter amphotericin B; Invitrogen, Carlsbad, Calif.), and0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). In some cases,different growth media are used or different supplementations areprovided, and these are normally indicated in the text assupplementations to growth medium. In certain chemically-defined mediathe cells may be grown without serum present at all. In such cases, thecells may require certain growth factors, which can be added to themedium to support and sustain the cells. Presently preferred factors tobe added for growth in serum-free media include one or more of bFGF,EGF, IGF-I, and PDGF. In more preferred embodiments, two, three or allfour of the factors are added to serum free or chemically defined media.In other embodiments, LIF is added to serum-free medium to support orimprove growth of the cells.

A “conditioned medium” is a medium in which a specific cell orpopulation of cells has been cultured, and then removed. When cells arecultured in a medium, they may secrete cellular factors that can providetrophic support to other cells. Such trophic factors include, but arenot limited to, hormones, cytokines, extracellular matrix (ECM),proteins, vesicles, antibodies, and granules. The medium containing thecellular factors is the conditioned medium.

Generally, a “trophic factor” is defined as a substance that promotessurvival, growth, differentiation, proliferation and/or maturation of acell, or stimulates increased activity of a cell.

When referring to cultured vertebrate cells, the term “senescence” (also“replicative senescence” or “cellular senescence”) refers to a propertyattributable to finite cell cultures, namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells, they are actually resistant to programmed cell death(apoptosis) and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

The term “neurological injury” is an inclusive term encompassingconditions associated with neuronal cell death or compromise, includingcerebrovascular insufficiency, focal or diffuse brain trauma, diffusebrain damage, and traumatic neuropathies (including, but not limited to,compression, crush, laceration and segmentation neuropathies). Examplesof neurological injury are: cerebral ischemia or infarction includingembolic occlusion, and thrombotic occlusion; reperfusion following acuteischemia; perinatal hypoxic-ischemic injury; cardiac arrest;intracranial hemorrhage of any type (such as epidural, subdural,subarachnoid and intracerebral); intracranial and intravertebral lesions(such as contusion, penetration, shear, compression and laceration);whiplash and shaken infant syndrome.

Other neurological injuries include tumors and other neoplasticconditions affecting the CNS and PNS. Though the underlying disease isconsidered proliferative (rather than an injury), surrounding tissuesmay be compromised. Furthermore, cell therapy may be utilized to deliverapoptotic or other antineoplastic molecules to the tumor site, e.g., viadelivery of genetically modified cells producing such agents.

The term “treating (or treatment of) a neurological injury” refers toameliorating the effects of, or delaying, halting or reversing theprogress of, or delaying or preventing the onset of, a neurologicalinjury as defined herein.

The term stimulating “regeneration capacity of a SVZ” refers toincreasing the ability of the subventricular zone to reform or remakesurrounding tissue, including neurological and endothelial tissue.

The term improving “neurological function” refers to making better afunction, such as, for example, muscle strength, reflex response, orsensory perception. Determination that the neurological function hasimproved is assessed by various behavioral tests and motor, sensory,reflex and balance tests.

The term “decreasing apoptosis in a damaged or injured part of apatient's brain” refers to lowering the number of cells undergoingapoptosis, or programmed cell death, in a part of a patient's brain thathas been subjected to some other injury or damage. The apoptosis can bedetermined by any methods known in the art, including but not limited toflow cytometry based apoptosis detection methods, immunohistochemistrymethods, DNA fragmentation assays, caspase activity assays and the like.The apoptosis can also be determined by in vitro assays known in the artto model or predict the response in vivo.

The term “effective amount” refers to a concentration or amount of acompound, material or composition as described herein, that is effectiveto achieve a particular biological result. Such results include, but arenot limited to cell growth and/or differentiation in vitro or in vivo,and treatment of a neurological injury as described herein. With respectto growth factors, an effective amount may range from about 1nanogram/ml to about 1 microgram/ml. With respect to UTC as administeredto a patient in vivo, an effective amount may range from as few asseveral hundred or fewer to as many as several million or more. Inspecific embodiments, an effective amount may range from about 10³ toabout 10¹¹ cells, more specifically at least about 10⁴ cells. It will beappreciated that the number of cells to be administered will varydepending on the specifics of the neurological injury to be treated,including but not limited to size or total volume/surface area to betreated, as well as proximity of the site of administration to thelocation of the region to be treated, among other factors familiar tothe medicinal biologist.

The terms “effective period,” “effective period of time” or “effectiveconditions” refer generally to a period of time or other controllableconditions (e.g., temperature, humidity for in vitro methods) necessaryor preferred for an agent or pharmaceutical composition to achieve itsintended result.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable medium,” which may be used interchangeably with the terms“biologically compatible carrier” or “biologically compatible medium,”refer to reagents, cells, compounds, materials, compositions, and/ordosage forms that are not only compatible with the cells and otheragents to be administered therapeutically, but also are suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other complicationcommensurate with a reasonable benefit/risk ratio. As described ingreater detail herein, pharmaceutically acceptable carriers suitable foruse in the present invention include liquids, semi-solid (e.g., gels)and solid materials (e.g., cell scaffolds and matrices, tubes, sheetsand other such materials as known in the art and described in greaterdetail herein). These semi-solid and solid materials may be designed toresist degradation within the body (non-biodegradable) or they may bedesigned to degrade within the body (biodegradable, bioerodable). Abiodegradable material may further be bioresorbable or bioabsorbable,i.e., it may be dissolved and absorbed into bodily fluids (water-solubleimplants are one example), or degraded and ultimately eliminated fromthe body, either by conversion into other materials or breakdown andelimination through natural pathways.

Several terms are used herein with respect to cell or tissuetransplantation or cell replacement therapy. The terms “autologoustransfer,” “autologous transplantation,” “autograft” and the like referto treatments wherein the cell or transplant donor is also the cell ortransplant recipient. The terms “allogeneic transfer,” “allogeneictransplantation,” “allograft” and the like refer to treatments whereinthe cell or transplant donor is of the same species as the recipient,but is not the same individual. A cell transfer in which the donor'scells have been histocompatibly matched with a recipient is sometimesreferred to as a “syngeneic transfer”. The terms “xenogeneic transfer,”“xenogeneic transplantation,” “xenograft” and the like refer totreatments wherein the cell or transplant donor is of a differentspecies than the recipient.

The term “isolate” as used herein generally refers to a cell which hasbeen separated from its natural environment. This term includes grossphysical separation from its natural environment, e.g., removal from thedonor animal. In preferred embodiments, an isolated cell is not presentin a tissue, i.e., the cell is separated or dissociated from theneighboring cells with which it is normally in contact. Preferably,cells are administered as a cell suspension. As used herein, the phrase“cell suspension” includes cells which are in contact with a medium andwhich have been dissociated, e.g., by subjecting a piece to tissue togentle trituration.

The term “matrix” as used herein generally refers to biodegradableand/or bioresorbable materials that are administered with the cells to apatient. The matrix may act as a temporary scaffold until replaced bynewly grown cells. In some embodiments, the matrix may provide for thesustained release of factors or other agents used in conjunction withthe cells and may provide a structure for developing tissue growth inthe patient. In other embodiments, the matrix simply provides atemporary scaffold for the developing tissue. The matrix can be inparticulate form (macroparticles greater than 10 microns in diameter ormicroparticles less than 10 micros in diameter), or it can be in theform of a structurally stable, three-dimensional implant (e.g., ascaffold). The matrix can be a slurry, hydrogel, or alternatively, athree dimensional structure such as a cube, cylinder, tube, block, film,sheet or an appropriate anatomical form.

The term “scaffold” as used herein generally refers to a threedimensional porous structure that provides a template for cell growth. Ascaffold is made of biodegradable and/or bioresorbable materials thatdegrade over time within the body. The length of time taken for thescaffold to degrade may depend upon the molecular weight of thematerials. Thus, higher molecular weight material may result in polymerscaffolds which retain their structural integrity for longer periods oftime; while lower molecular weights results in both slower release andshorter scaffold lives. The scaffold may be made by any means known inthe art. Examples of polymers which can be used to form the scaffoldinclude natural and synthetic polymers.

Description

Neurological injuries, which encompass conditions associated withneuronal cell death or compromise, including cerebrovascularinsufficiency, focal or diffuse brain trauma, diffuse brain damage, andtraumatic neuropathies, have as a common feature the dysfunction or lossof a specific or vulnerable group of neural cells. This commonalityenables development of similar therapeutic approaches for the repair andregeneration of vulnerable or damaged neural tissue, one of which iscell-based therapy. In its various embodiments described herein, thepresent invention features methods and pharmaceutical compositions forneural repair and regeneration that utilize progenitor cells and cellpopulations derived from postpartum umbilical cord tissues. Theinvention is applicable to any neurological injury, but is expected tobe particularly suitable for a number of injuries including, withoutlimitation, cerebral ischemia or infarction including embolic occlusionand thrombotic occlusion, reperfusion following acute ischemia,perinatal hypoxic-ischemic injury, cardiac arrest, as well asintracranial hemorrhage of any type (such as epidural, subdural,subarachnoid and intracerebral), and intracranial and intravertebrallesions (such as contusion, penetration, shear, compression andlaceration), and also whiplash and shaken infant syndrome.

As summarized above, the invention, in one of its aspects is generallydirected to methods of treating neurological injuries using isolatedumbilical cord tissue-derived cells (UTC), which are derived fromumbilical cord tissue that has been rendered substantially free ofblood. The UTC is capable of self-renewal and expansion in culture andhas the potential to differentiate into cells of neural phenotypes.Certain embodiments feature populations comprising such cells,pharmaceutical compositions comprising the cells or components orproducts thereof, and methods of using the pharmaceutical compositionsfor treatment of patients with neurological injuries. The umbilical cordtissue-derived cells have been characterized by their growth propertiesin culture, by their cell surface markers, by their gene expression, bytheir ability to produce certain biochemical trophic factors, and bytheir immunological properties.

According to the methods described herein, mammalian umbilical cordtissue is digested and the UTC is isolated preferably in an asepticenvironment. Blood and debris are preferably removed from the postpartumtissue prior to isolation of the UTC. For example, the postpartum tissuemay be washed with buffer solution, such as but not limited to phosphatebuffered saline. The wash buffer also may comprise one or moreantimycotic and/or antibiotic agents, such as, but not limited to,penicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Whole tissue or a fragment or section thereof may be disaggregated bymechanical force (mincing or shear forces). In a presently preferredembodiment, the isolation procedure also utilizes an enzymatic digestionprocess. Many enzymes are known in the art to be useful for theisolation of individual cells from complex tissue matrices to facilitategrowth in culture. Ranging from weakly digestive (e.g.deoxyribonucleases and the neutral protease, dispase) to stronglydigestive (e.g. papain and trypsin), such enzymes are availablecommercially. A nonexhaustive list of such enzymes includes mucolyticenzyme activities, metalloproteases, neutral proteases, serine proteases(such as trypsin, chymotrypsin, or elastase), and deoxyribonucleases.Presently preferred are enzyme activities selected frommetalloproteases, neutral proteases and mucolytic activities. Forexample, collagenases are known to be useful for isolating various cellsfrom tissues. Deoxyribonucleases can digest single-stranded DNA and canminimize cell-clumping during isolation. Preferred methods involveenzymatic treatment with collagenase and dispase, or collagenase,dispase, and hyaluronidase. The skilled artisan will appreciate thatmany such enzyme treatments are known in the art for isolating cellsfrom various tissue sources, and is well-equipped to assess new, oradditional enzymes or enzyme combinations for their utility in isolatingthe cells of the invention. Preferred enzyme treatments can be fromabout 0.5 to 2 hours long or longer. In other preferred embodiments, thetissue in incubated at about 37° C. during the enzyme treatment of thedissociation step.

The isolated cells may be used to initiate, or seed, cell cultures.Isolated cells are transferred to sterile tissue culture vessels eitheruncoated or coated with extracellular matrix or ligands such as laminin,collagen (native, denatured or crosslinked), gelatin, fibronectin, andother extracellular matrix proteins. The cells are cultured in anyculture medium capable of sustaining growth of the cells such as, butnot limited to, DMEM (high or low glucose), advanced DMEM, DMEM/MCDB201, Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium(F12), Iscove's modified Dulbecco's medium, mesenchymal stem cell growthmedium (MSCGM), DMEM/F12, RPMI 1640, and CELL-GRO-FREE. The culturemedium may be supplemented with one or more components including, forexample, fetal bovine serum (FBS), preferably about 2-15% (v/v); equineserum (ES); human serum (HS); beta-mercaptoethanol (BME or 2-ME),preferably about 0.001% (v/v); one or more growth factors, for example,platelet-derived growth factor (PDGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitoryfactor (LIF) and erythropoietin; amino acids, including L-valine; andone or more antibiotic and/or antimycotic agents to control microbialcontamination, such as, for example, penicillin G, streptomycin sulfate,amphotericin B, gentamicin, and nystatin, either alone or incombination. The culture medium preferably comprises growth medium (e.g.DMEM-low glucose, serum, BME, and an antibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. Preferably, the cells are cultured at about 0 to about 5 percentCO₂ by volume in air and at about 2 to about 25 percent O₂ by volume inair, preferably about 5 to about 20 percent O₂ in air. The cellspreferably are cultured at about 25° C. to about 40° C. and morepreferably are cultured at 37° C. The cells are preferably cultured inan incubator. The medium in the culture vessel can be static oragitated, for example, using a bioreactor. The UTC preferably is grownunder low oxidative stress (e.g., with addition of glutathione, vitaminC, catalase, vitamin E, N-acetylcysteine). “Low oxidative stress,” asused herein, refers to conditions of no or minimal free radical damageto the cultured cells.

In some embodiments of the invention, the UTC may be passaged or removedto a separate culture vessel containing fresh medium of the same or adifferent type as that used initially, where the population of cells canbe mitotically expanded. Cells useful in the methods of the inventionmay be used at any point between passage 0 and senescence. The cellspreferably are passaged between about 3 and about 25 times, morepreferably are passaged about 4 to about 12 times, and preferably arepassaged 10 or 11 times. Cloning and/or subcloning may be performed toconfirm that a clonal population of cells has been isolated.

Further, the different cell types present in postpartum tissue may befractionated into subpopulations from which the UTC can be isolated.This may be accomplished using standard techniques for cell separationincluding, but not limited to, enzymatic treatment to dissociatepostpartum tissue into its component cells, followed by cloning andselection of specific cell types, including, but not limited to:selection based on morphological and/or biochemical markers; selectivegrowth of desired cells (positive selection); selective destruction ofunwanted cells (negative selection); separation based upon differentialcell agglutinability in the mixed population as, for example, withsoybean agglutinin; freeze-thaw procedures; differential adherenceproperties of the cells in the mixed population; filtration;conventional and zonal centrifugation; centrifugal elutriation(counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and fluorescence activatedcell sorting (FACS).

The culture medium is changed as necessary. Incubation is continueduntil a sufficient number or density of cells accumulate in the dish.Thereafter, any original explanted tissue sections that exist may beremoved, and the remaining cells separated from the dish bytrypsinization using standard techniques or by using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to be theUTC.

The UTC may be cryopreserved. Accordingly, UTC for autologous transfer(for either the mother or child) may be derived from appropriatepostpartum tissues following the birth of a child, then cryopreserved soas to be available in the event they are later needed fortransplantation.

The UTC may be characterized, for example, by growth characteristics(e.g., population doubling capability, doubling time, passages tosenescence), karyotype analysis (e.g., normal karyotype; maternal orneonatal lineage), flow cytometry (e.g., FACS analysis),immunohistochemistry and/or immunocytochemistry (e.g., for detection ofepitopes), gene expression profiling (e.g., gene chip arrays; polymerasechain reaction (for example, reverse transcriptase PCR, real time PCR,and conventional PCR)), protein arrays, protein secretion (e.g., byplasma clotting assay or analysis of UTC-conditioned medium, forexample, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocytereaction (e.g., as measure of stimulation of PBMCs), and/or othermethods known in the art.

Examples of umbilicus tissue-derived cells were deposited with theAmerican Type Culture Collection on Jun. 10, 2004, and assigned ATCCAccession Numbers as follows: (1) strain designation UMB 022803 (P7) wasassigned Accession No. PTA-6067; and (2) strain designation UMB 022803(P17) was assigned Accession No. PTA-6068.

The UTC useful in the methods of the invention may possess one or moreof the following growth features: (1) they require L-valine for growthin culture; (2) they are capable of growth in atmospheres containingoxygen from about 5% to about 20%; (3) they have the potential for atleast about 40 doublings in culture before reaching senescence; and (4)they attach and expand on tissue culture vessels that are uncoated, orthat are coated with gelatin, laminin, collagen, polyornithine,vitronectin or fibronectin.

Additionally, the UTC useful in the methods of the invention may possessa normal karyotype, which is maintained as the cells are passaged.Methods for karyotyping are available and known to those of skill in theart.

Also, the UTC useful in the methods of the invention may becharacterized by production of certain proteins, including: (1)production of at least one of tissue factor, vimentin, and alpha-smoothmuscle actin; and (2) production of at least one of: CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A, B, C cell surface markers, asdetected by flow cytometry. Additionally, the UTC useful in the methodsof the invention may be characterized by lack of production of at leastone of: CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G,and HLA-DR, DP, DQ cell surface markers, as detected by flow cytometry.UTC useful in the methods of the invention may produce at least two of:tissue factor; vimentin; and alpha-smooth muscle actin; or all three ofthe proteins tissue factor, vimentin, and alpha-smooth muscle actin.

Further, the UTC useful in the methods of the invention may becharacterized by gene expression, which relative to a human cell that isa fibroblast, a mesenchymal stem cell, or an iliac crest bone marrowcell, is increased for a gene encoding at least one of: interleukin 8;reticulon 1; chemokine (C-X-C motif) ligand 1 (melonoma growthstimulating activity, alpha); chemokine (C-X-C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C-X-C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3.

Also, the UTC useful in the methods of the invention may becharacterized by gene expression, which relative to a human cell that isa fibroblast, a mesenchymal stem cell, or an iliac crest bone marrowcell, is reduced for a gene encoding at least one of: short staturehomeobox 2; heat shock 27 kDa protein 2; chemokine (C-X-C motif) ligand12 (stromal cell-derived factor 1); elastin (supravalvular aorticstenosis, Williams-Beuren syndrome); Homo sapiens mRNA; cDNADKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeo box 2 (growtharrest-specific homeo box); sine oculis homeobox homolog 1 (Drosophila);crystallin, alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA F1112280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein F1120373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin,beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA fulllength insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein F1114054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDainteracting protein 3-like; AE binding protein 1; and cytochrome coxidase subunit VIIa polypeptide 1 (muscle).

Additionally, the UTC useful in the methods of the invention may becharacterized by secretion of at least one of: MCP-1; IL-6; IL-8; GCP-2;HGF; KGF; FGF; HB-EGF; BDNF; TPO; MIPb; I309; MDC; RANTES; and TIMP1.Further, the UTC useful in the methods of the invention may becharacterized by lack of secretion of at least one of: TGF-beta2; ANG2;PDGFbb; MIP1a; and VEGF, as detected by ELISA.

The UTC useful in the methods of the invention preferably comprise twoor more of the above-listed growth, protein/surface marker production,gene expression or substance-secretion characteristics. The UTC usefulin the methods of the invention may comprise three, four, five, six,seven, eight or more of the characteristics. The UTC useful in themethods of the invention may also comprise all of above characteristics.

Among the UTC useful in the methods of the invention in several of itsaspects is the UTC having the characteristics described above and moreparticularly those wherein the cells have normal karyotypes and maintainnormal karyotypes with passaging, and further wherein the cells expresseach of the markers CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A, B, C, wherein the cells produce the immunologically-detectableproteins which correspond to the listed markers. The UTC useful in themethods of the invention may also include, in addition to the foregoing,cells that do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD117, CD141, or HLA-DR, DP, DQ, as detected by flowcytometry.

Certain cells having the potential to differentiate along lines leadingto various phenotypes are unstable and thus can spontaneouslydifferentiate. The UTC useful in the methods of the invention are cellsthat do not spontaneously differentiate, for example, along neurallines. The UTC useful in the methods of the invention, when grown ingrowth medium, are substantially stable with respect to the cell markersproduced on their surface, and with respect to the expression pattern ofvarious genes, for example, as determined using an Affymetrix GENECHIP.The cells remain substantially constant, for example, in their surfacemarker characteristics over passaging, and through multiple populationdoublings.

However, one feature of the UTC useful in the methods of the inventionis that they may be deliberately induced to differentiate into neurallineage phenotypes by subjecting them to differentiation-inducing cellculture conditions. This may be accomplished by one or more methodsknown in the art. For instance, as exemplified herein, the UTC may beplated on flasks coated with laminin in Neurobasal-A medium (Invitrogen,Carlsbad, Calif.) containing B27 (B27 supplement, Invitrogen),L-glutamine and Penicillin/Streptomycin, the combination of which isreferred to herein as Neural Progenitor Expansion (NPE) medium. NPEmedia may be further supplemented with bFGF and/or EGF. Alternatively,the UTC useful in the methods of the invention may be induced todifferentiate in vitro by (1) co-culturing the UTC with neuralprogenitor cells, or (2) growing the UTC in neural progenitorcell-conditioned medium.

Differentiation of the UTC may be demonstrated by a bipolar cellmorphology with extended processes. The induced cell populations maystain positive for the presence of nestin. The differentiated UTC may beassessed by detection of nestin, TuJ1 (BIII tubulin), GFAP, tyrosinehydroxylase, GABA, O4 and/or MBP. Additionally, the UTC useful in themethods of the invention may exhibit the ability to formthree-dimensional bodies characteristic of neuronal stem cell formationof neurospheres.

The UTC useful in the methods of the invention may include a cellpopulation that is heterogeneous. A heterogeneous cell population usefulin the methods of the invention may comprise at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% UTC as described above.The heterogeneous cell populations useful in the methods of theinvention may further comprise stem cells or other progenitor cells,such as neural progenitor cells, or may further comprise fullydifferentiated neural cells. Additionally, the population may besubstantially homogeneous, i.e., comprises substantially only the UTC(such as at least about 96%, 97%, 98%, 99% or more UTC). The homogeneouscell population useful in the methods of the invention may compriseumbilicus- or placenta-derived cells. Homogeneous populations ofumbilicus-derived cells are preferably free of cells of maternallineage. Homogeneous populations of placenta-derived cells may be ofneonatal or maternal lineage. Homogeneity of a cell population may beachieved by any method known in the art, for example, by cell sorting(e.g., flow cytometry) or by clonal expansion in accordance with knownmethods. Thus, homogeneous UTC populations useful in the methods of theinvention may comprise a clonal cell line of umbilical cordtissue-derived cells. Such populations are particularly useful when acell clone with highly desirable functionality has been isolated.

Additionally, the UTC useful in the methods of the invention may includepopulations of cells incubated in the presence of one or more factors,or under conditions, that stimulate stem cell differentiation along aneurogenic pathway. Such factors are known in the art and the skilledartisan will appreciate that determination of suitable conditions fordifferentiation can be accomplished with routine experimentation.Optimization of such conditions can be accomplished by statisticalexperimental design and analysis, for example response surfacemethodology allows simultaneous optimization of multiple variables, forexample in a biological culture. Exemplary factors include, but are notlimited to factors, such as growth or trophic factors, demethylatingagents, co-culture with neural lineage cells or culture in neurallineage cell-conditioned medium, as well other conditions known in theart to stimulate stem cell differentiation along a neurogenic pathway orlineage. (See, e.g., Lang, K J D, et al., J. Neurosci. Res., 2004;76:184-192; Johe, KK, et al., Genes Devel., 1996; 10:3129-3140;Gottleib, D, Ann. Rev. Neurosci., 2002; 25:381-407).

The UTC useful in the methods of the invention may also be geneticallymodified to produce neurotherapeutically useful gene products, or toproduce antineoplastic agents for treatment of tumors, for example.Genetic modification may be accomplished using any of a variety ofvectors including, but not limited to, integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors; non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Othermethods of introducing DNA into cells include the use of liposomes,electroporation, a particle gun, or by direct DNA injection.

Hosts cells may be transformed or transfected with DNA controlled by, orin operative association with, one or more appropriate expressioncontrol elements such as promoter or enhancer sequences, transcriptionterminators, polyadenylation sites, among others, and a selectablemarker. Any promoter may be used to drive the expression of the insertedgene. For example, viral promoters include, but are not limited to, theCMV promoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus orelastin gene promoter. Additionally, the control elements used tocontrol expression of the gene of interest can allow for the regulatedexpression of the gene so that the product is synthesized only whenneeded in vivo. If transient expression is desired, constitutivepromoters may be used in a non-integrating and/or replication-defectivevector. Alternatively, inducible promoters could be used to drive theexpression of the inserted gene when necessary. Inducible promotersinclude, but are not limited to, those associated with metallothioneinand heat shock proteins.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines. This methodcan be advantageously used to engineer cell lines that express the geneproduct.

The UTC useful in the methods of the invention may be geneticallyengineered to “knock out” or “knock down” expression of factors thatpromote inflammation or rejection at the implant site. Negativemodulatory techniques for the reduction of target gene expression levelsor target gene product activity levels are discussed below. “Negativemodulation,” as used herein, refers to a reduction in the level and/oractivity of target gene product relative to the level and/or activity ofthe target gene product in the absence of the modulatory treatment. Theexpression of a gene native to a neuron or glial cell can be reduced orknocked out using a number of techniques including, for example,inhibition of expression by inactivating the gene using the homologousrecombination technique. Typically, an exon encoding an important regionof the protein (or an exon 5′ to that region) is interrupted by apositive selectable marker, e.g., neo, preventing the production ofnormal mRNA from the target gene and resulting in inactivation of thegene. A gene may also be inactivated by creating a deletion in part of agene, or by deleting the entire gene. By using a construct with tworegions of homology to the target gene that are far apart in the genome,the sequences intervening the two regions can be deleted. (Mombaerts etal., Proc. Nat. Acad. Sci. U.S.A., 1991; 88:3084). Antisense, DNAzymes,ribozymes, small interfering RNA (siRNA) and other such molecules thatinhibit expression of the target gene can also be used to reduce thelevel of target gene activity. For example, antisense RNA molecules thatinhibit the expression of major histocompatibility gene complexes (HLA)have been shown to be most versatile with respect to immune responses.Still further, triple helix molecules can be utilized in reducing thelevel of target gene activity. These techniques are described in detailby Davis, L G, et al., (eds), Basic Methods in Molecular Biology, 2nded., 1994, Appleton & Lange, Norwalk, Conn.

Additionally, cell lysates and cell soluble fractions prepared from theUTC, or heterogeneous or homogeneous cell populations comprising UTC, aswell as the UTC or populations thereof that have been geneticallymodified or that have been stimulated to differentiate along aneurogenic pathway, which are useful in the methods of the invention,are provided. Use of the UTC lysate soluble fraction (i.e.,substantially free of membranes) in vivo, for example, allows thebeneficial intracellular milieu to be used allogeneically in a patientwithout introducing an appreciable amount of the cell surface proteinsmost likely to trigger rejection, or other adverse immunologicalresponses. Methods of lysing cells are well-known in the art and includevarious means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their growth medium, and thus containsecreted growth factors and the like, or they may be prepared from cellswashed free of medium in, for example, PBS or other solution. Washedcells may be resuspended at concentrations greater than the originalpopulation density if preferred.

Whole cell lysates of the UTC useful in the methods of the invention maybe prepared, e.g., by disrupting cells without subsequent separation ofcell fractions. Alternatively, a cell membrane fraction may be separatedfrom a soluble fraction of the cells by routine methods known in theart, e.g., centrifugation, filtration, or similar methods.

Cell lysates or cell soluble fractions prepared from populations ofumbilical cord tissue-derived cells useful in the methods of theinvention may be used as is, further concentrated by, for example,ultrafiltration or lyophilization, or even dried, partially purified,combined with pharmaceutically-acceptable carriers or diluents as areknown in the art, or combined with other compounds such as biologicals,for example, pharmaceutically useful protein compositions. Cell lysatesor fractions thereof may be used in vitro or in vivo, alone or, forexample, with autologous or syngeneic live cells. The lysates, ifintroduced in vivo, may be introduced locally at a site of treatment, orremotely to provide, for example, needed cellular growth factors to apatient.

Additionally, the UTC useful in the methods of the invention may becultured in vitro to produce biological products in high yield. Forexample, such cells, which either naturally produce a particularbiological product of interest (e.g., a trophic factor), or that havebeen genetically engineered to produce a biological product, can beclonally expanded using the culture techniques described herein.Alternatively, cells may be expanded in a medium that inducesdifferentiation to a neural lineage or other lineage. In either case,biological products produced by the cell and secreted into the mediumcan be readily isolated from the conditioned medium using standardseparation techniques, e.g., such as differential protein precipitation,ion-exchange chromatography, gel filtration chromatography,electrophoresis, and HPLC, to name a few. A “bioreactor” may be used totake advantage of the flow method for feeding, for example, athree-dimensional culture in vitro. Essentially, as fresh media ispassed through the three-dimensional culture, the biological product iswashed out of the culture and may then be isolated from the outflow, asabove.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells be lysed, asdescribed above. The biological product may then be purified using anyone or more of the above-listed techniques.

Additionally, conditioned medium from the cultured UTC useful in themethods of the invention may be used in vitro and in vivo as describedbelow. Use of the UTC conditioned medium allows the beneficial trophicfactors secreted by the UTC to be used allogeneically in a patientwithout introducing intact cells that could trigger rejection, or otheradverse immunological responses. Conditioned medium is prepared byculturing cells in a culture medium, then removing the cells from themedium.

Conditioned medium prepared from populations of the UTC useful in themethods of the invention may be used as is, further concentrated, by forexample, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically-acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Conditioned medium may be used in vitro or in vivo, alone or forexample, with autologous or syngeneic live cells. The conditionedmedium, if introduced in vivo, may be introduced locally at a site oftreatment, or remotely to provide, for example needed cellular growth ortrophic factors to a patient.

Additionally, an extracellular matrix (ECM) produced by culturing theUTC useful in the methods of the invention on liquid, solid orsemi-solid substrates may be prepared, collected and utilized as analternative to implanting live cells into a subject in need of tissuerepair or replacement. The UTC is cultured in vitro, on a threedimensional framework as described elsewhere herein, under conditionssuch that a desired amount of ECM is secreted onto the framework. Thecells comprising the new tissue are removed, and the ECM processed forfurther use, for example, as an injectable preparation. To accomplishthis, cells on the framework are killed and any cellular debris isremoved from the framework. This process may be carried out in a numberof different ways. For example, the living tissue can be flash-frozen inliquid nitrogen without a cryopreservative, or the tissue can beimmersed in sterile distilled water so that the cells burst in responseto osmotic pressure.

Once the cells have been killed, the cellular membranes may be disruptedand cellular debris removed by treatment with a mild detergent rinse,such as EDTA, CHAPS or a zwitterionic detergent. Alternatively, thetissue can be enzymatically digested and/or extracted with reagents thatbreak down cellular membranes and allow removal of cell contents.Examples of such enzymes include, but are not limited to, hyaluronidase,dispase, proteases, and nucleases. Examples of detergents includenon-ionic detergents such as, for example, alkylaryl polyether alcohol(TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and HaasPhiladelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl ether (AtlasChemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), apolyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylenelauryl ether (Rohm and Haas); and ionic detergents such as, for example,sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonatedalkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in abranched or unbranched chain.

The collection of the ECM can be accomplished in a variety of ways,depending, for example, on whether the new tissue has been formed on athree-dimensional framework that is biodegradable or non-biodegradable.For example, if the framework is non-biodegradable, the ECM can beremoved by subjecting the framework to sonication, high pressure waterjets, mechanical scraping, or mild treatment with detergents or enzymes,or any combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM.

After it has been collected, the ECM may be processed further. Forexample, the ECM can be homogenized to fine particles using techniqueswell known in the art such as by sonication, so that it can pass througha surgical needle. The components of the ECM can be crosslinked, ifdesired, by gamma irradiation. For example, the ECM can be irradiatedbetween 0.25 to 2 mega rads to sterilize and crosslink the ECM. Chemicalcrosslinking using agents that are toxic, such as glutaraldehyde, ispossible but not generally preferred.

The amounts and/or ratios of proteins, such as the various types ofcollagen present in the ECM, may be adjusted by mixing the ECM producedby the UTC useful in the methods of the invention with ECM of one ormore other cell types. In addition, biologically active substances suchas proteins, growth factors and/or drugs, can be incorporated into theECM. Exemplary biologically active substances include tissue growthfactors, such as TGF-beta, and the like, which promote healing andtissue repair at the site of the injection. Such additional agents maybe utilized with, for example, whole cell lysates, soluble cellfractions, or further purified components and products produced by theUTC.

In another aspect, the invention provides pharmaceutical compositionsthat utilize the UTC, UTC populations, components and products of theUTC in various methods for treating neurological injury, improvingneurological function, stimulating the regenerative capacity of the SVZor decreasing apoptosis in the SVZ. Some pharmaceutical compositionscomprise live cells (UTC alone or admixed with other cell types). Otherpharmaceutical compositions comprise UTC cellular components (e.g., celllysates, soluble cell fractions, conditioned medium, ECM, or componentsof any of the foregoing) or products (e.g., trophic and other biologicalfactors produced naturally by the UTC or through genetic modification,conditioned medium from UTC culture). In any case, the pharmaceuticalcomposition may further comprise other active agents, such asanti-inflammatory agents, anti-apoptotic agents, antioxidants, growthfactors, neurotrophic factors or neuroregenerative or neuroprotectivedrugs as known in the art.

Examples of other components that may be added to the UTC pharmaceuticalcompositions include, but are not limited to: (1) other neuroprotectiveor neurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, the UTC may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (7) local anesthetics,to name a few.

Pharmaceutical compositions encompassed by the invention comprise UTC,or components or products thereof, formulated with a pharmaceuticallyacceptable carrier or medium. Suitable pharmaceutically acceptablecarriers include water, salt solution (such as Ringer's solution),alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose,or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrolidine. Such preparations can be sterilized, and if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, and salts for influencing osmotic pressure,buffers, and coloring. Pharmaceutical carriers suitable for use in thepresent invention are known in the art and are described, for example,in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton, Pa.) andWO 96/05309.

Typically, but not exclusively, pharmaceutical compositions comprisingUTC components or products, but not live cells, are formulated asliquids (or as solid tablets, capsules and the like, when oral deliveryis appropriate). These may be formulated for administration by anyacceptable route known in the art to achieve delivery of drugs andbiological molecules to the target neural tissue, including, but notlimited to, oral, nasal, ophthalmic and parenteral, includingintravenous. Particular routes of parenteral administration include, butare not limited to, intramuscular, subcutaneous, intraperitoneal,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

Pharmaceutical compositions comprising the live UTC cells are typicallyformulated as liquids, semisolids (e.g., gels) or solids (e.g.,matrices, scaffolds and the like, as appropriate for neural tissueengineering). Liquid compositions are formulated for administration byany acceptable route known in the art to achieve delivery of live cellsto the target neural tissues. Typically, these include injection orinfusion into the CNS or PNS, either in a diffuse fashion or targeted tothe site of neurological injury or distress, by a route ofadministration including, but not limited to, intraocular,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are typically formulated for surgical implantation at thesite of neurological injury or distress. It will be appreciated thatliquid compositions also may be administered by surgical procedures.Additionally, semi-solid or solid pharmaceutical compositions maycomprise semi-permeable gels, lattices, cellular scaffolds and the like,which may be non-biodegradable or biodegradable. For example, it may bedesirable or appropriate to sequester the exogenous cells from theirsurroundings, yet enable the cells to secrete and deliver biologicalmolecules (e.g. neurotrophic factors) to surrounding neural cells.Cells, therefore, may be formulated as autonomous implants comprisingliving UTC or a cell population comprising UTC surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression.

Alternatively, different varieties of degradable gels and networks areutilized for the pharmaceutical compositions of the invention. Forexample, degradable materials particularly suitable for sustainedrelease formulations include biocompatible polymers, such as poly(lacticacid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like.

Additionally, it may be desirable or appropriate to deliver the cells onor in a biodegradable, preferably bioresorbable or bioabsorbable,scaffold or matrix. These typically three-dimensional biomaterialscontain the living cells attached to the scaffold, dispersed within thescaffold, or incorporated in an extracellular matrix entrapped in thescaffold. Once implanted into the target region of the body, theseimplants become integrated with the host tissue, wherein thetransplanted cells gradually become established. (See, e.g., Tresco, PA, et al., Adv. Drug Delivery Rev., 2000; 42:3-27; see also Hutmacher, DW, J. Biomater. Sci. Polymer Edn., 2001; 12:107-174).

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe trade name VICRYL (Ethicon, Inc., Somerville, N.J.), Foams, composedof, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying orlyophilizing, as discussed in U.S. Pat. No. 6,355,699 also may beutilized. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. In situ-forming degradable networks are also suitable foruse in the invention (see, e.g., Anseth, K S, et al., J. ControlledRelease, 2002; 78:199-209; Wang, D, et al., Biomaterials, 2003;24:3969-3980; U.S. Patent Publication 2002/0022676). These materials areformulated as fluids suitable for injection, then may be induced by avariety of means (e.g., change in temperature, pH, exposure to light) toform degradable hydrogel networks in situ or in vivo.

Also, the framework may be a felt, which can be composed of amultifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures.

Further, the framework may be molded into a useful shape, such as thatof the spinal cord with segregated columns for nerve tract repair, forexample (Friedman, J A, et al., Neurosurgery, 2002; 51:742-51).Furthermore, it will be appreciated that the UTC may be cultured onpre-formed, non-degradable surgical or implantable devices, e.g., in amanner corresponding to that used for preparing fibroblast-containingGDC endovascular coils, for instance (Marx, W F, et al., Am. J.Neuroradiol., 2001; 22:323-333).

The matrix, scaffold or device may be treated prior to the inoculationof cells to enhance cell attachment. For example, prior to inoculation,nylon matrices can be treated with 0.1 molar acetic acid and incubatedin polylysine, PBS, and/or collagen to coat the nylon. Polystyrene canbe similarly treated using sulfuric acid. The external surfaces of aframework may also be modified to improve the attachment or growth ofcells and differentiation of tissue, such as by plasma coating theframework or addition of one or more proteins (e.g., collagens, elasticfibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g.,heparin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatansulfate, keratin sulfate), a cellular matrix, and/or other materialssuch as, but not limited to, gelatin, alginates, agar, agarose, andplant gums, among others.

The UTC-containing frameworks are prepared according to methods known inthe art. For example, cells can be grown freely in a culture vessel tosub-confluency or confluency, lifted from the culture and inoculatedonto the framework. Growth factors may be added to the culture mediumprior to, during, or subsequent to inoculation of the cells to triggerdifferentiation and tissue formation, if desired. Alternatively, theframeworks themselves may be modified so that the growth of cellsthereon is enhanced, or so that the risk of rejection of the implant isreduced. Thus, one or more biologically active compounds, including, butnot limited to, anti-inflammatories, immunosuppressants or growthfactors, may be added to the framework for local release.

The UTC, or cell populations comprising UTC, or components of orproducts produced by the UTC, may be used in a variety of ways tosupport and facilitate repair and regeneration of neural cells andtissues. Such utilities encompass in vitro, ex vivo and in vivo methods.

In Vitro and Ex Vivo Methods:

The UTC may be used in vitro to screen a wide variety of compounds foreffectiveness and cytotoxicity of pharmaceutical agents, growth factors,regulatory factors, and the like. For example, such screening may beperformed on substantially homogeneous populations of the UTC to assessthe efficacy or toxicity of candidate compounds to be formulated with,or co-administered with, the UTC, for treatment of a neurologicalinjury. Alternatively, such screening may be performed on the UTC thathas been stimulated to differentiate into a neural cell or neuralprogenitor cell, for the purpose of evaluating the efficacy of newpharmaceutical drug candidates. In this embodiment, the UTC aremaintained in vitro and exposed to the compound to be tested. Theactivity of a potentially cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth or regulatory factorsmay be assessed by analyzing the number or robustness of the culturedcells, as compared with cells not exposed to the factors. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens.

Additionally, as discussed above, the UTC can be cultured in vitro toproduce biological products that are either naturally produced by thecells, or produced by the cells when induced to differentiate intoneural or other lineages, or produced by the cells via geneticmodification. For instance, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF,MIP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were found to be secretedfrom UTC grown in growth medium. Some of these trophic factors, such asBDNF and IL-6, have important roles in neural regeneration. Othertrophic factors, as yet undetected or unexamined, of use in neuralrepair and regeneration, are likely to be produced by the UTC andpossibly secreted into the medium.

Also, the UTC may be used for production of conditioned medium, eitherfrom the undifferentiated UTC or from the UTC incubated under conditionsthat stimulate differentiation into a neural or other lineage. Suchconditioned media are contemplated for use in in vitro or ex vivoculture of neurogenic precursor cells, or in vivo to supporttransplanted cells comprising homogeneous populations of the UTC orheterogeneous populations comprising UTC and neural progenitors, forexample.

Additionally, UTC lysates, soluble cell fractions or components thereof,or ECM or components thereof, may be used for a variety of purposes. Asmentioned above, some of these components may be used in pharmaceuticalcompositions. Also, a cell lysate or ECM may be used to coat orotherwise treat substances or devices to be used surgically, or forimplantation, or for ex vivo purposes, to promote healing or survival ofcells or tissues contacted in the course of such treatments.

Further, the UTC may be used in co-cultures in vitro to provide trophicsupport to other cells, in particular neural cells and neuralprogenitors. For co-culture, it may be desirable for the UTC and thedesired other cells to be co-cultured under conditions in which the twocell types are in contact. This can be achieved, for example, by seedingthe cells as a heterogeneous population of cells in culture medium oronto a suitable culture substrate. Alternatively, the UTC can first begrown to confluence, and then will serve as a substrate for the seconddesired cell type in culture. Additionally, the cells may further bephysically separated, e.g., by a membrane or similar device, such thatthe other cell type may be removed and used separately, following theco-culture period. Use of the UTC in co-culture to promote expansion anddifferentiation of neural cell types may find applicability in researchand in clinical/therapeutic areas. For instance, the UTC co-culture maybe utilized to facilitate growth and differentiation of neural cells inculture, for basic research purposes or for use in drug screeningassays, for example. The UTC co-culture may also be utilized for ex vivoexpansion of neural progenitors for later administration for therapeuticpurposes. For example, neural progenitor cells may be harvested from anindividual, expanded ex vivo in co-culture with the UTC, then returnedto that individual (autologous transfer) or another individual(syngeneic or allogeneic transfer). Following ex vivo expansion, themixed population of cells comprising the UTC and neural progenitors maybe administered to a patient in need of treatment. Alternatively, insituations where autologous transfer is appropriate or desirable, theco-cultured cell populations may be physically separated in culture,enabling removal of the autologous neural progenitors for administrationto the patient.

In Vivo Methods:

As set forth in Examples 2-10, UTC have been shown to be effectivelytransplanted into the body, and to supply lost neural function in ananimal model accepted for its predictability of efficacy in humans. Oncetransplanted into a targeted neural location in the body, the UTC maythemselves differentiate into one or more neural phenotypes, or they mayprovide trophic support for neural progenitors and neural cells in situ,or they may exert a beneficial effect in both of those fashions, as wellas others.

The UTC may be administered alone (e.g., as substantially homogeneouspopulations) or as admixtures with other cells. As described above, theUTC may be administered as formulated in a pharmaceutical preparationwith a matrix or scaffold, or with conventional pharmaceuticallyacceptable carriers. Where the UTC are administered with other cells,they may be administered simultaneously or sequentially with the othercells (either before or after the other cells). Cells that may beadministered in conjunction with the UTC include, but are not limitedto, neurons, astrocytes, oligodendrocytes, neural progenitor cells,neural stem cells and/or other multipotent or pluripotent stem cells.The cells of different types may be admixed with the UTC immediately orshortly prior to administration, or they may be co-cultured together fora period of time prior to administration.

The UTC may be administered with other neuro-beneficial drugs orbiological molecules, or other active agents, such as anti-inflammatoryagents, anti-apoptotic agents, antioxidants, growth factors,neurotrophic factors or neuroregenerative or neuroprotective drugs asknown in the art. When the UTC are administered with other agents, theymay be administered together in a single pharmaceutical composition, orin separate pharmaceutical compositions, simultaneously or sequentiallywith the other agents (either before or after administration of theother agents).

Examples of other components that may be administered with the UTCinclude, but are not limited to: (1) other neuroprotective orneurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, UTC may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics,to name a few.

For example, the UTC may be administered as undifferentiated cells,i.e., as cultured in growth medium. Alternatively, the UTC may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired neural phenotype, e.g., astrocyte,oligodendrocyte or neuron, and more specifically, serotoninergic,dopaminergic, cholinergic, GABA-ergic or glutamatergic neurons (see,e.g., Isacson, O, Lancet Neurology, 2003; 2(7):417-424, or other lineagethat supports neural regeneration or repair.

The UTC may be surgically implanted, injected, delivered (e.g., by wayof a catheter or syringe), or otherwise administered directly orindirectly to the site of neurological damage or distress. Routes ofadministration of the UTC or compositions thereof include, but are notlimited to, intravenous, intramuscular, subcutaneous, intranasal,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. Liquid or fluid pharmaceutical compositions,however, may be administered to a more general location in the CNS orPNS (e.g., throughout a diffusely affected area, such as would be thecase in diffuse ischemic injury, for example), inasmuch as neuralprogenitor cells have been shown to be capable of extensive migrationfrom a point of entry to the nervous system to a particular location,e.g., by following radial glia or by responding to chemical signals.

This migratory ability of neural stem cells has opened a new avenue fortreatment of malignant brain tumors, i.e., use of progenitor cells fordelivery of therapeutic genes/gene products for the treatment of thesemigratory tumors. For example, it has been reported that neural stemcells, when implanted into intracranial gliomas in vivo in adultrodents, distribute themselves quickly and extensively through the tumorbed and migrate in juxtaposition to expanding and advancing tumor cells,while continuing to stably express a foreign gene (Aboody, K, et al.,Proc. Natl. Acad. Sci. USA, 2000; 97:12846-12851). The UTC are alsoexpected to be suitable for this type of use, i.e., the UTC geneticallymodified to produce an apoptotic or other antineoplastic agent, e.g.,IL-12 (Ehtesham, M, et al., Cancer Research, 2002; 62:5657-5663) ortumor necrosis factor-related apoptosis-inducing ligand (Ehtesham, M, etal., Cancer Research, 2002; 62:7170-7174) may be injected or otherwiseadministered to a general site of a malignant tumor (e.g.,glioblastoma), whereafter the UTC can migrate to the tumor cells forlocal delivery of the therapeutic agent. The UTC can also facilitateneurological repair following tumor treatment, as described above, bydifferentiation into one or more neural phenotypes, or by providingtrophic support for neural progenitors and neural cells.

Additionally, methods of treating neurological injury by administeringpharmaceutical compositions comprising the UTC cellular components(e.g., cell lysates or components thereof) or products (e.g., trophicand other biological factors produced naturally by the UTC or throughgenetic modification, conditioned medium from the UTC culture) areprovided by the invention. Again, these methods may further compriseadministering other active agents, such as growth factors, neurotrophicfactors or neuroregenerative or neuroprotective drugs as known in theart.

Dosage forms and regimes for administering the UTC or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the neurodegenerativecondition, age, sex, body weight and general medical condition, andother factors known to medical practitioners. Thus, the effective amountof a pharmaceutical composition to be administered to a patient isdetermined by these considerations as known in the art.

Because the CNS is a somewhat immunoprivileged tissue, it may not benecessary or desirable to immunosuppress a patient prior to initiationof cell therapy with the UTC. Previously, it has been shown that UTC donot stimulate allogeneic PBMCs in a mixed lymphocyte reaction. (See,U.S. patent application Ser. No. 10/877,269, which issued as U.S. Pat.No. 7,524,489). Accordingly, transplantation with allogeneic, or evenxenogeneic, UTC may be tolerated in some instances.

In other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device, as described above. These and othermeans for reducing or eliminating an immune response to the transplantedcells are known in the art. As an alternative, the UTC may begenetically modified to reduce their immunogenicity, as mentioned above.

Survival of transplanted UTC in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the neural tissue, andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for neural cells or productsthereof, e.g., neurotransmitters. Transplanted cells can also beidentified by prior incorporation of tracer dyes such as rhodamine- orfluorescein-labeled microspheres, fast blue, ferric microparticles,bisbenzamide or genetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted UTC into neural tissue of asubject can be assessed by examining restoration of the neural functionthat was damaged or diseased. Such functions include, but are notlimited to motor, cognitive, sensory and endocrine functions, inaccordance with procedures well known to neurobiologists and physicians.This restoration of neural function by the UTC can be used in methods toimprove neurological function in patients following neurological injury.

Additionally, the UTC may be used in methods of stimulating theregenerative capacity of the SVZ in patients. For example, theregenerative capacity of the SVZ may be stimulated by showing that thereis an increase in neurogenesis, angiogenesis or synaptogenesis. Anincrease in neurogenesis indicates that progenitor cells in the SVZ areproliferating in preparation to replace injured or damaged neural cellsand that there are newly formed neuroblasts and other immature neurons.An increase in angiogenesis indicates that new blood vessel formation isoccurring in the injured or damaged area to provide an oxygen supply tothe injured or damaged tissue or to tissue that is being formed toreplace the injured or damaged tissue. An increase in synaptogenesisindicates that new synapses are being formed, most likely in response tosome stimulus that caused a decrease in the number of functioningsynapses already present. The ability of the UTC to cause increases inneurogenesis, angiogenesis and synaptogenesis are set forth in Examples3-10.

Further, the UTC may decrease the number of apoptotic cells in theinjured or damaged part of the brain. Apoptosis may be a cause ofsecondary brain injury following traumatic brain injury and high ratesof apoptosis may be associated with poorer prognosis after traumaticbrain injury. (See, Minambres, et al., Journal of Neurotrauma, 2008;25(6):581-591). A decrease in apoptosis in an area of the brain that hasexperienced injury or damage, therefore, may increase survival, improveneurological function and recovery from injury, and may act as anadjunct to other therapies, such as stimulating the regenerativecapacity of the SVZ in patients following injury. Examples 7 and 9demonstrate the ability of the UTC to decrease apoptosis in the damagedsections of brain tissue.

In another aspect, the invention provides kits that utilize the UTC, UTCpopulations, components and products of the UTC in various methods forneural regeneration and repair as described above. Where used fortreatment of neurological injury, or other scheduled treatment, the kitsmay include one or more cell populations, including at least UTC and apharmaceutically acceptable carrier (liquid, semi-solid or solid). Thekits also optionally may include a means of administering the cells, forexample by injection. The kits further may include instructions for useof the cells. Kits prepared for field hospital use, such as for militaryuse may include full-procedure supplies including tissue scaffolds,surgical sutures, and the like, where the cells are to be used inconjunction with repair of acute injuries. Kits for assays and in vitromethods as described herein may contain one or more of (1) the UTC orcomponents or products of the UTC, (2) reagents for practicing the invitro method, (3) other cells or cell populations, as appropriate, and(4) instructions for conducting the in vitro method.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

The following abbreviations may appear in the examples and elsewhere inthe specification and claims:

-   -   ANG2 (or Ang2) for angiopoietin 2    -   APC for antigen-presenting cells    -   BDNF for brain-derived neurotrophic factor    -   bFGF for basic fibroblast growth factor    -   bid (BID) for “bis in die” (twice per day)    -   CK18 for cytokeratin 18    -   CNS for central nervous system    -   CXC ligand 3 for chemokine receptor ligand 3    -   DMEM for Dulbecco's Minimal Essential Medium    -   DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose    -   EDTA for ethylene diamine tetraacetic acid    -   EGF (or E) for epidermal growth factor    -   FACS for fluorescent activated cell sorting    -   FBS for fetal bovine serum    -   FGF (or F) for fibroblast growth factor    -   GCP-2 for granulocyte chemotactic protein-2    -   GFAP for glial fibrillary acidic protein    -   HB-EGF for heparin-binding epidermal growth factor    -   HCAEC for Human coronary artery endothelial cells    -   HGF for hepatocyte growth factor    -   hMSC for Human mesenchymal stem cells    -   HNF-1alpha for hepatocyte-specific transcription factor 1 alpha;        HUVEC for Human umbilical vein endothelial cells    -   I309 for a chemokine and the ligand for the CCR8 receptor    -   IGF-1 for insulin-like growth factor 1    -   IL-6 for interleukin-6; IL-8 for interleukin 8    -   K19 for keratin 19; K8 for keratin 8    -   KGF for keratinocyte growth factor    -   LIF for leukemia inhibitory factor    -   MBP for myelin basic protein    -   MCP-1 for monocyte chemotactic protein 1    -   MDC for macrophage-derived chemokine    -   MIP1alpha for macrophage inflammatory protein 1 alpha    -   MIP1beta for macrophage inflammatory protein 1 beta    -   MMP for matrix metalloprotease (MMP)    -   MSC for mesenchymal stem cells    -   NHDF for Normal Human Dermal Fibroblasts    -   NPE for Neural Progenitor Expansion media    -   O4 for oligodendrocyte or glial differentiation marker O4    -   PBMC for Peripheral blood mononuclear cell    -   PBS for phosphate buffered saline    -   PDGFbb for platelet derived growth factor    -   PO for “per os” (by mouth)    -   PNS for peripheral nervous system    -   Rantes (or RANTES) for regulated on activation, normal T cell        expressed and secreted    -   rhGDF-5 for recombinant human growth and differentiation factor        5    -   SC for subcutaneously    -   SDF-1alpha for stromal-derived factor 1 alpha    -   SHH for sonic hedgehog    -   SOP for standard operating procedure    -   TARC for thymus and activation-regulated chemokine    -   TCP for Tissue culture plastic    -   TCPS for tissue culture polystyrene    -   TGFbeta2 for transforming growth factor beta2    -   TGF beta-3 for transforming growth factor beta-3    -   TIMP1 for tissue inhibitor of matrix metalloproteinase 1    -   TPO for thrombopoietin    -   TuJ1 for BIII Tubulin    -   VEGF for vascular endothelial growth factor    -   vWF for von Willebrand factor    -   alphaFP for alpha-fetoprotein.

Additionally, as used in the following examples and elsewhere in thespecification, the UTC useful in the methods of the invention may beisolated and characterized according to the disclosure of U.S. patentapplication Ser. No. 10/877,269, which is incorporated by reference inits entirety as it relates to the description, isolation andcharacterization of UTC.

EXAMPLE 1 Lone-Term Neural Differentiation of Cells

The ability of umbilicus-derived cells to undergo long-termdifferentiation into neural lineage cells was evaluated. The UTC wereisolated and expanded as described in Examples 13-15.

Frozen aliquots of UTC (umbilicus (022803) P11; (042203) P11; (071003)P12) previously grown in growth medium were thawed and plated at 5,000cells/cm² in T-75 flasks coated with laminin (BD, Franklin Lakes, N.J.)in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27(B27 supplement, Invitrogen), L-glutamine (4 mM), andPenicillin/Streptomycin (10 milliliters), the combination of which isherein referred to as Neural Progenitor Expansion (NPE) media. NPE mediawas further supplemented with bFGF (20 ng/ml, Peprotech, Rocky Hill,N.J.) and EGF (20 ng/ml, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

In addition, adult human dermal fibroblasts (P11, Cambrex, Walkersville,Md.) and mesenchymal stem cells (P5, Cambrex) were thawed and plated atthe same cell seeding density on laminin-coated T-75 flasks inNPE+bFGF+EGF. As a further control, fibroblasts, umbilicus, andplacenta-derived cells were grown in growth medium for the periodspecified for all cultures.

Media from all cultures were replaced with fresh media once a week andcells observed for expansion. In general, each culture was passaged onetime over a period of one month because of limited growth inNPE+bFGF+EGF.

After a period of one month, all flasks were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature.Immunocytochemistry was performed using antibodies directed against TuJ1(BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) and GFAP (glial fibrillaryacidic protein; 1:2000; DakoCytomation, Carpinteria, Calif.). Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing block along with goat anti-mouse IgG—Texas Red (1:250;Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488(1:250; Molecular Probes). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

TABLE 1-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, Mo. GFAP 1:2000DakoCytomation, Carpinteria, Ca.

Immediately following plating, a subset of UTC attached to the cultureflasks coated with laminin. This may have been due to cell death as afunction of the freeze/thaw process or because of the new growthconditions. Cells that did attach adopted morphologies different fromthose observed in growth media.

Upon confluence, cultures were passaged and observed for growth. Verylittle expansion took place of those cells that survived passage. Atthis point, very small cells with no spread morphology and withphase-bright characteristics began to appear in cultures ofumbilicus-derived cells. These areas of the flask were followed overtime. From these small cells, bifurcating processes emerged withvaricosities along their lengths, features very similar to previouslydescribed PSA-NCAM+ neuronal progenitors and TuJ1+ immature neuronsderived from brain and spinal cord (Mayer-Proschel, M, et al. Neuron,1997; 19(4):773-85; Yang, H, et al., PNAS, 2000; 97(24):13366-71). Withtime, these cells became more numerous, yet still were only found inclones. This indicates that NPE+bFGF+EGF media slows proliferation ofPPDCs and alters their morphology.

Cultures were fixed at one month post-thawing/plating and stained forthe neuronal protein TuJ1 and GFAP, an intermediate filament found inastrocytes. While all control cultures grown in growth medium and humanfibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to beTuJ1−/GFAP−, TuJ1 was detected in the umbilicus and placenta PPDCs.Expression was observed in cells with and without neuronal-likemorphologies. No expression of GFAP was observed in either culture. Thepercentage of cells expressing TuJ1 with neuronal-like morphologies wasless than or equal to 1% of the total population (n=3 umbilicus-derivedcell isolates tested). While not quantified, the percentage of TuJ1+cells without neuronal morphologies was higher in umbilicus-derived cellcultures than placenta-derived cell cultures. These results appearedspecific as age-matched controls in growth medium did not express TuJ1.These results indicate that clones of UTC express neuronal proteins.

Methods for generating differentiated neurons (based on TuJ1 expressionand neuronal morphology) from UTC were developed. While expression forTuJ1 was not examined earlier than one month in vitro, it is clear thatat least a small population of UTC can give rise to neurons eitherthrough default differentiation or through long-term induction followingone month's exposure to a minimal media supplemented with L-glutamine,basic FGF, and EGF.

EXAMPLE 2 Effect of Administration of Cells Following Brain Injury onNeurological Function. Intracerebral Hemorrhage (ICH) Model in Rat

ICH was induced in male Wistar rats weighing 300-350 g by injecting 100μl of autologous blood as essentially described by Seyfried, et al., J.Neurosurg, 2006; 104:313-318 (2006). Briefly, rats were anesthetizedwith xylazine (10 mg/kg) and ketamine (80 mg/kg). Once adequateanesthesia was achieved, the rats were maintained at 37° C. throughoutthe surgical procedure using a feedback regulated water heating pad.Under a dissecting scope, a 2 cm ventral skin incision was made alongthe crease formed by the abdomen and right thigh. Blunt dissection ofthe adductor muscles is used to visualize the right femoral artery. Fiveto ten millimeters of artery was carefully mobilized from the adjacentfemoral vein and saphenous nerve. The artery was ligated at the distalend, and the proximal portion was temporarily blocked with a 4-0 suture.A PESO catheter was then inserted into the vessel 1-2 cm through a smallpuncture and secured in place with a 4-0 suture. Then the rat was placedprone on a stereotactic frame (David Kopf Instruments, Tujunja, Calif.).A midline incision was made over the calvarium and carried down to theperiosteum. A small periosteal elevator was used to expose the skull.Once this was accomplished, the stereotactic frame measurements wereused to guide the site where the craniectomy took place. Firstidentification of the bregma was performed and then a craniectomy wasperformed with the stereotactic drill (3.5 mm lateral to midline, 0.5 mmanterior to bregma, depth 5.5 mm below the surface to midline). 0.3 mlof autologous blood taken from the femoral artery was placed into a 1 ccsyringe with a 26G½ needle that was loaded on the stereotactic frame.0.1 ml of blood was then infused at a rate of 10 μl per minute with aninfusion pump (600-910/920, Harvard Apparatus, Holliston, Mass.). Toprevent blood from backing up, a piece of bone wax was used to close thecraniectomy site and keep the blood in the brain. The skin wasreapproximated with 4.0 silk suture, simple running.

At 24 hours or 72 hours after ICH, randomly selected animals underwentcell transplantation. Animals were anesthetized with 3.5% halothane inN₂O:O₂ (2:1) and maintained at 0.5% halothane using a facemask. UTC(see, U.S. patent application Ser. No. 10/877,269 for a description ofisolation and characterization of UTCs useful in the methods of theinvention) in 2 ml total fluid volume PBS or PBS alone (control) wereinjected into a tail vein of the animal. Experimental groups (n=8/group)consisted of PBS alone and UTC (3×10⁶). All rats were allowed to survive28 days after surgery. In all animals, batteries of behavioral testswere measured one day, four days, and weekly thereafter.

mNSS is a composite of motor, sensory, balance and reflex tests.Neurological function, as assessed by mNSS (as described by Chen, J, etal., Stroke, 2001; 32:1005-1011) was graded on a scale of 0 to 18(normal score 0; maximal deficit score 18). In the severity scores ofinjury, one point is awarded for the exhibition of certain abnormalbehavior or for the lack of a tested reflex. Thus, the higher score, themore severe is the injury. The motor tests used were raising the rat bythe tail, for which the following scores were given: flexion of forelimb(1), flexion of hindlimb (1), head moved more than 10° to the verticalaxis within 30 seconds (1); and, walking on the floor, for which thefollowing scores were given: normal walk (0), inability to walk straight(1), circling toward the paretic side (2), falling down to the pareticside (3). The sensory tests used were a placing test (visual and tactiletest) and a proprioceptive test (deep sensation, pushing the paw againstthe table edge to stimulate limb muscles). The balance test used was abalance beam test, for which the following scores were given: balanceswith steady posture (0), grasps side of beam (1), hugs the beam and onelimb falls down from the beam (2), hugs the beam and two limbs fall downfrom the beam, or spins on beam (>60 s) (3), attempts to balance on thebeam but falls off (>40 s) (4), attempts to balance on the beam butfalls off (>20 s) (5), and falls off, with no attempt to balance or hangon to the beam (<20 s) (6).

The corner test, as described by Zhang, L, et al., J Neurosci Methods,2002; 117(2):207-14, was performed. Briefly, a rat was placed betweentwo attached boards (dimensions of 30×20×1 cm3). The edges of the twoboards were at a 30° angle with a small opening along the joint toencourage entry into the corner. The rat was placed facing and half wayto the corner. When entering deep into the corner, both sides of thevibrissae were stimulated together. The rat then reared forward andupward, and then turned back to face the open end. A non-injured rateither turned left or right, but the injured rats preferentially turnedtoward the non-impaired side. The turns in one versus the otherdirection were recorded from ten trials for each test, and the fractionof the turns was used as the corner test score.

Results from the functional tests indicated that rats treated 24 hoursand 72 hours with UTC (3×10⁶) exhibited significant (p<0.05) improvementin the mNSS test and corner test. (See, FIGS. 1 and 2). mNSS totalscores decreased significantly among rats treated at 24 hour and 72 hourwith 3×10⁶ UCT as compared to controls, at days 14, 21, and 28. (See,FIG. 1).

EXAMPLE 3 Effect of Administration of Cells Following Brain Injury onCell Proliferation in the Subventricular Zone

Bromodeoxyuridine (BrdU), a thymidine analog, can be incorporated intocells' genomic DNA during the S phase of cell cycle. BrdU positive cellsin the subventricular zone (SVZ) are considered to be progenitor cellsundergoing DNA synthesis in the S phase of the cell cycle. The number ofBrdU cells in the SVZ is used as an indicator of neurogenesis.

The rats received daily intraperitoneal injections (IP) of 100 mg/Kg ofBrdU starting at 24 hours after ICH and subsequently for the next 14days. After 28 days, animals were reanesthetized with ketamine (80mg/kg) and (xylazine 13 mg/kg) IP injection, and sacrificed (first bydraining all of the blood from the body via a heart puncture andflushing the system with normal saline and then 4% paraformaldehyde).The skull was then removed with a rongeur and the brain removed andsubsequently fixed in 4% paraformaldehyde and sliced/sectioned togrossly and histologically assess the region of the hemorrhage.Immunohistochemical staining was used for BrdU (mouse monoclonalantibody, 1:100; Boehringer Mannheim, Indianapolis, Ind.). Briefly, thebrain tissue residing between +0.1 and 0.86 mm of the bregma on thethird block was the most severely injured and therefore the third blockwas specifically selected for immunostaining. Every 40th coronal sectionfrom +0.1-0.86 mm of the bregma was used for immunochemical stainingwith the same antibody. Sections were blocked in a Tris-buffered salinecontaining 5% normal goat serum, 1% BSA and 0.05% TWEEN-20. Sectionswere then incubated with the primary antibodies following with theincubation with the appropriate secondary antibodies. Controlexperiments consisted of staining brain coronal tissue sections asoutlined above, but omitted the primary antibodies. BrdU-positive cellnumbers in the ipsilateral subventricular zone (SVZ) were counted withuse of an Olympus BX40 microscope and a 3-CCD color video camera (SonyDXC-970MD) interfaced with an MCID image analysis system (ImagingResearch, St. Catharines, Canada). The total numbers of BrdU-positivecells in the ipsilateral SVZ are reported.

The results show that the number of BrdU positive cells weresignificantly increased in the SVZ of the ipsilateral hemisphere of ratstreated with 3×10⁶ UTC at 24 hours or 72 hours after ICH compared withthe control PBS group (p<0.05) (See FIGS. 3E and 3F).

EXAMPLE 4 Effect of Administration of Cells Following Brain Injury onAngiogenesis in the Damaged Area of the Brain

Enlarged and thin-walled vessels in the boundary around the lesion areindicative of angiogenesis (Li, Y, et al., Neurology, 2002; 59:514-523).After 28 days, animals were reanesthetized, sacrificed and their brainsremoved as detailed in Example 3 for histological assessment. Amonoclonal antibody against vWF (1:400, Dako, Carpinteria, Calif.) wasused.

The results show that the perimeters of vessels were significantlyincreased in treatment groups with 3×10⁶ UTC at 24 hours or 72 hoursafter ICH compared with the control PBS group (p<0.05) (See FIGS. 4E and4F).

Additionally, as noted in Example 3 above, BrdU can be incorporated intocells' genomic DNA during the S phase of cell cycle. By assessing theexpression of Von Willebrand Factor in conjunction with BrdUincorporation it can be determined if actively dividing cells arecontributing to the growth of new blood vessels at the ICH boundary (seeprotocol above).

Endothelial cells positive for BrdU are shown in FIG. 5A. Vesselspositive for Von Willebrand Factor (vWF) are shown in FIG. 5B. FIG. 5Cshows double immunostaining of BrdU reactive cells colocalized with vWFpositively stained tissues in the vessel. Double staining revealed asubpopulation of cells that express a vascular marker while stilldividing, suggesting that cells positive for vascular phenotype arenewly formed during the recovery stage.

EXAMPLE 5 Effect of Administration of Cells Following Brain Injury onNeurogenesis in the Subventricular Zone

Doublecortin (DCX) is a marker of neurogenesis, which is transientlyexpressed (during about 2-3 weeks) in newly formed neuroblasts. After 28days, animals were reanesthetized, sacrificed and their brains removedas detailed in Example 3 for histological assessment. Goat anti-DCX(1:200; Santa Cruz Biotechnology, Santa Cruz, Calif.) was used.

The results show immunoexpression for DCX in SVZ is significantlyincreased in treatment groups with 3×10⁶ UTC at 24 hours or 72 hoursafter ICH compared with the control PBS group (p<0.05). (See FIGS. 6Eand 6F).

Additionally, BIII Tubulin (TUJ1) is a neuron specific tubulin that isexpressed during fetal and postnatal development and in putativeneuronal cells of the SVZ. TUJ1 expression was assessed to detect anynewly formed immature neurons. A mouse anti-TUJ1 (1:1000, NovusBiologicals Inc. Littleton, Colo.) was used.

The results show immunoexpression for TUJ1 in SVZ is significantlyincreased in treatment groups with 3×10⁶ UTC at 24 hours or 72 hoursafter ICH compared with the control PBS group (p<0.05). (See FIGS. 7Eand 7F).

EXAMPLE 6 Effect of Administration of Cells Following Brain Injury onSynaptogenesis in the Boundary Zone of Hematoma

Synaptophysin, a presynaptic vesicle protein, is used as an indicator ofsynaptogenesis (Ujike, H, et al., Ann N Y Acad Sci., 2002; 965:55-67).After 28 days, animals were reanesthetized, sacrificed and their brainsremoved as detailed in Example 3 for histological assessment.Synaptophysin (1:40 mAb, Clone SY 38, Millipore, Billerica, Mass.) wasused.

The results show that the expression of synaptophysin increasedsignificantly along the boundary zone of hematoma with 3×10⁶ UTC at 24hours or 72 hours after ICH compared with the control PBS group.(p<0.05.). (See FIGS. 8E and 8F).

EXAMPLE 7 Effect of Administration of Cells Following Brain Injury onApoptosis in the Damaged Area of the Brain

After 28 days, animals were reanesthetized, sacrificed and their brainsremoved as detailed in Example 3 for histological assessment. Theterminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-endlabeling (TUNEL) method (ApopTag Kit; Oncor, Gaithersburg, Md.) was usedto assess in situ apoptotic detection according to manufacturer'sspecifications. The TUNEL method is based on the specific binding of TdTto 3′-OH ends of DNA and the ensuing synthesis of polydeoxynucleotidepolymer cells. Briefly, after deparaffinizing brain sections anddigesting protein using proteinase K and then quenching endogenousperoxidase activity with 2% H₂O₂ in PBS, the slides were placed inequilibration buffer and then in working-strength TdT enzyme, followedby working-strength stop/wash buffer. After two drops ofantidigoxigenin-peroxidase were applied to the slides, peroxidase wasdetected with DAB. Negative controls were performed with distilled waterfor TdT enzyme in the preparation of working-strength TdT. The labelingtarget of the TUNEL method was the new 3′-OH DNA ends generated by DNAfragmentation, which were typically localized in morphologicallyidentifiable nuclei and apoptotic bodies with dark brown, rounded oroval bodies.

The TUNEL staining showed apoptotic cells with typical dark brown,rounded or oval apoptotic bodies. Scattered apoptotic cells were presentthroughout the damaged tissue, the vast majority of which was located inthe boundary zone of hematoma. The results show that the number ofapoptotic cells were significantly reduced in the ipsilateral hemispherewith 3×10⁶ UTC at 24 hours or 72 hours after ICH compared with thecontrol PBS group. (p<0.05). (See FIGS. 9E and 9F).

EXAMPLE 8 Effect of Administration of Cells Following Brain Injury onTissue Loss in the Damaged Area of the Brain

After 28 days, animals were reanesthetized, sacrificed and their brainsremoved as detailed in Example 3 for histological assessment. Thecerebral tissues were cut into 7 equally spaced (2 mm) coronal blocks,and then processed for paraffin sectioning. A series of adjacent6-μm-thick sections were cut from each block in the coronal plane andwere stained with hematoxylin and eosin. The sections were traced by aGlobal Laboratory Image analysis system (Data Translation, Mariborn,Mass.). The area of preserved striatum on the side of the hemorrhage wassubtracted from that of the contralateral side, thus determining thedegree of tissue loss attributable to the ICH, and comparing treated tountreated animals. The results show that there is no significantdifference in tissue loss with 3×10⁶ UTC at 24 hours or 72 hours afterICH compared with the control PBS group. (See FIGS. 10A and 10B).

EXAMPLE 9 Effect of Administration of Cells at Different Time PointsFollowing Brain Injury

Examples 2-8 describe the effect of administration of UTC at 24 hoursand 72 hours post-injury. In the present study, the therapeutic windowwas expanded by a delayed administration at 7 days, which better mimicsthe clinical situation (i.e., more patients will be able to be treatedif the cells could be administered at 7 days).

ICH was induced in adult Wistar rats as described in Example 2 (SeeSeyfried, D, et al., J Neurosurg, 2006; 104:313-318), with a group ofrats (n=40) divided into 4 groups of 10 animals each. At 72 hours or 1week after ICH, randomly selected animals underwent celltransplantation. Animals were anesthetized with 3.5% halothane in N₂O:O₂(2:1) and maintained at 0.5% halothane using a facemask. UTC in 2 mltotal fluid volume PBS or PBS alone (control) were injected into a tailvein. Experimental groups (n=10/group) consisted of PBS alone and UTC (3million). All rats were allowed to survive 28 days after celltransplantation.

In all animals, batteries of behavioral tests were measured one day, 4days, 1 week, 2 weeks, 3 weeks, 4 weeks, 31 days and 35 days thereafter.

As described in Example 2, nMSS (See Chen, J, et al., Stroke, 2001;32:1005-1011) and the Corner Test (see Zhang, L, et al., J NeurosciMethods, 2002; 117:207-214) indicate neurological function.

The Cylinder Test (see Zhang, L, et al., J Neurosci Methods, 2002;117:207-214, and Hua, Y, et al., Stroke, 2002; 33:2478-2484) was adaptedfor use in rat to assess forelimb use and rotation asymmetry in atransparent cylinder (20 in cm diameter and 30 cm in height) for 3 to 10minutes depending on the degree of activity during the trial. A mirrorwas placed to the side of the cylinder at an angle to enable therecording of forelimb movements even when the animal was turned awayfrom the camera. Scoring was done by an experimenter blinded to thecondition of the animal using a video cassette recorder with slow-motionand clear stop-frame capabilities. The behavior was scored according tothe following criteria: (1) independent use of the left or rightforelimb for contacting the wall during a full rear to initiate aweight-shifting movement or to regain center of gravity while movinglaterally in a vertical posture and (2) simultaneous use of both theleft and right forelimbs for contacting the cylinder wall during a fullrear and for alternating lateral stepping movements along the wall.

For the Adhesive-Removal Somatosensory Test (see, Chen, J, et al.,Stroke, 2001; 32:2682-2688) small pieces of adhesive-backed paper dots(of equal sizes, 56.55 mm²) were used as bilateral tactile stimulioccupying the distal-radial region on the wrist of each forelimb. Therat was then returned to its cage. The time to remove each stimulus fromforelimbs was recorded on 5 trials per day. Before surgery, the animalswere trained for 3 days. Once the rats were able to remove the dotswithin 10 seconds, they were subjected to ICH.

Results

1. Neurological Functional

Results from the functional tests indicated that rats treated at day 7or day 3 with 3×10⁶ UTC exhibited significant (p<0.05) neurologicalfunctional improvement in the mNSS test, corner test, and cylinder test.

mNSS: mNSS total scores decreased significantly at days 21, 28, and 35in rats receiving treatment at 7 days; and total scores decreasedsignificantly at days 14, 21, 28 and 31 among rats treated at day 3 with3×10⁶ UTC compared to controls. (See, FIGS. 11A and 11B).

Corner Test: Corner test scores decreased significantly at days 21 and28; and scores decreased but not significantly at days 31 and 35 amongrats treated at day 7 or day 3 with 3×10⁶ UTC compared to controls.(See, FIGS. 12A and 12B).

Cylinder Test: Cylinder scores decreased significantly at days 21, 28,31 and 35, among rats treated at day 7 or day 3 with 3×10⁶ UTC comparedto controls. (See, FIGS. 13A and 13B).

Adhesive Test: Adhesive test scores did not decrease significantly atany time point, among rats treated at day 7 or day 3 with 3×10⁶ UTCcompared to controls. (See, FIGS. 14A and 14B).

2. Histology

The number of BrdU positive cells were significantly increased in theSVZ of the ipsilateral hemisphere of rats treated with 3×10⁶ UTC at day3 or day 7 after ICH compared with the control PBS group (p<0.05). (See,FIGS. 15A-15F).

Enlarged and thin-walled vessels in the boundary around the lesion areindicative of angiogenesis (See, Li, Y, et al., Neurology, 2002;59:514-523). Data showed that the perimeters of vessels weresignificantly increased in treatment groups with 3×10⁶ UTC at day 7 orday 3 after ICH compared with the control PBS group (p<0.05). (See,FIGS. 16A-16F).

BrdU-positive endothelial cells and vWF-positive vessels are shown atthe ICH boundary in FIGS. 17A-17D. FIGS. 17E and 17F show doubleimmunostaining of BrdU reactive cells colocalized with vWF positivelystained tissues in the vessel. Double staining revealed a subpopulationof cells that express a vascular marker while still dividing, suggestingthat cells positive for vascular phenotype are newly formed during therecovery stage. (See, FIGS. 17E-17F).

TUJ1 labeling was performed to detect any newly formed immature neurons.In this study, immunoexpression for TUJ1 in SVZ is significantlyincreased in treatment groups with 3×10⁶ UTC cells at day 7 or day 3after ICH compared with the control PBS group (p<0.05). (See, FIGS.18A-18 F).

Synaptophysin, a presynaptic vesicle protein, is used as an indicator ofsynaptogenesis (See, Ujike, H, et al., Ann N Y Acad Sci., 2002;965:55-67). Data demonstrated that the expression of synaptophysinincreased significantly along the boundary zone of hematoma in the UTCcell treatment groups compared with the control PBS group (p<0.05).(See, FIGS. 19A-19F).

TUNEL staining showed apoptotic cells in the brain with typical darkbrown, rounded or oval apoptotic bodies. Scattered apoptotic cells werepresent throughout the damaged tissue, the vast majority of which waslocated in the boundary zone of hematoma. Apoptotic cells aresignificantly reduced in the ipsilateral hemisphere in the UTC treatmentgroups at day 3 after ICH compared with the control PBS group (p<0.05).No significant differences of the total apoptotic cells number weredetected among day 7 groups. (See, FIGS. 20A-20F).

To determine tissue loss, cerebral tissues were cut into 7 equallyspaced (2 mm) coronal blocks, and then processed for paraffinsectioning. A series of adjacent 6-um-thick sections were cut from eachblock in the coronal plane and were stained with hematoxylin and eosin.The sections were traced by the Global Laboratory Image analysis system(Data Translation, Marlboro, Mass.). The area of preserved striatum onthe side of the hemorrhage was subtracted from that of the contralateralside, thus reckoning the degree of tissue loss from the ICH, andcomparing treated to untreated animals. The results show that there isno significant difference in tissue loss with 3×10⁶ UTC at 3 days or 7days after ICH compared with the control PBS group. (See, FIGS. 21A and21B).

For donor cell identification, the grafted human cells in the brain ofrats were stained with three different antibodies: NuMa (Ab-2) Mouse mAb(107-7) (Anti-Nuclear Matrix, Catalog# NA09L, Calbiochem); PurifiedMouse Anti-Human β2-Microglobulin (Catalog#555550, BD Biosciences); andMouse anti-Human Mitochondria (Catalog#E5204, Spring Bioscience Corp).The immunostaining was performed at the same time with two negativecontrols (i.e., the omission of primary antibody and the use ofpre-immune serum) and one positive control for quality control of theimmunoassay procedure. The positive control was human cells that weretransplanted into the brains of mice. The result showed that thepositive control worked very well, but no positive stained cells wereseen in our experimental slides and negative control slides. We suspectthat there are factors either with the tissue processing or the batch ofinjected cells that caused no reaction with the antibodies.

The results show that the transplanted UTC administrated at day 7 or day3 after ICH can significantly improve functional outcomes by mNSS test,the corner turn test and cylinder test in treatment groups compared withthe control PBS group (P<0.05). The treatment effects becamestatistically significant at day 14 after ICH, and persisted at leastuntil 28 days after surgery. In addition, significantly more BrdUpositive cells and cells with TUJ1 expression are presented in the SVZof ipsilateral hemisphere of rats in treatment groups with 3×10⁶ UTCgiven at day 7 or day 3 after ICH compared with the control PBS group(p<0.05). Microvessels and synaptophysin expression were significantlyincreased in the boundary zone of the injured area; and significantlylower numbers of apoptotic cells were found in the treatment group withinjected cells at day 3 after ICH compared with the control group. Thebeneficial effects of intravenous infusion of UTC were not significantlydifferent between day 7 and day 3 administrations after ICH.

EXAMPLE 10 Effect of Administration of Cells Following Brain Injury toEnhance Tissue Repair

UTC and MSCs were tested to treat rats with traumatic brain injury toseek a new way to enhance tissue repair. UTC or MSCs were administeredto treat young adult male rats after TBI.

24 male Wistar rats, body weight 300-330 g were used for this experiment(Charles River Breeding Company). After an appropriate period ofquarantine, each rat was anesthetized with chloral hydrate (400 mg/kg).Buprenex 0.05 mg/kg was given preoperatively to all animals. The ratswere subjected to controlled cortical impact (CCI). Body temperature wasmaintained at 37° C. with a heated pad and K module.

24 rats were divided in three groups (8 per group). Two treatment groupsreceived UTC or MSCs (4×10⁶ in 2 ml of PBS) administered through thetail vein 24 h after TBI. For this rats were anesthetized with chloralhydrate 400 mg/kg administered intraperitoneally (i.p). Control animalsreceived only 2 ml PBS, i.v.

All rats were tested on modified neurological severity score (mNSS) testand Morris Water Maze test at different time points after TBI. Forlabeling proliferating cells, bromodeoxyuridine (BrdU, 200 mg/kg; SigmaChemical) was injected (intraperitoneally) daily for 14 days into ratsstarting 1 day after TBI. All rats were euthanized 35 days after TBI byinjecting ketamine (160 mg/kg) and xylazine (20 mg/kg) i.p. The braintissue was processed for histological analysis (staining).

mNSS was performed 1 day before TBI and then on days 1, 4, 7, 14, 28 and35 after TBI. Morris Water Maze tests were performed days 31-35 afterTBI. Data collection was automated by the HVS Image 2020 Plus TrackingSystem (US HVS Image, San Diego, Calif.).

All brain samples were stained with standard H&E as well asimmunohistochemistry. H&E staining was performed to calculate lesionvolume. Immunohistochemistry was done for identification of UTC or MSCsusing anti-human mitochondrial antibody (E 5204).

To identify newly proliferating cells BrdU immunohistochemistry wasdone. Double staining with MAP-2 and vWF was performed to identify thephenotype of newly proliferating cells.

Results

There were significant differences in mNSS between both treated groups(UTC or MSCs treated rats) and the control group which was first visibleat day 7 and persisted until the end of trial. (See, FIG. 22). There wasno difference between the two treated groups.

There was improvement seen on day 35 in the UTC or MSCs treated groupscompared to the control with Morris Water Maze test. (See, FIG. 23)

For Lesion volume calculation, there was no significant difference inlesion volume between UTC or MSC treated rats and the control group ofanimals. (See, FIG. 24) (*P=0.07, ^(#)P=0.2)

To identify UTC or MSCs, immunohistochemistry was performed using E5204antibody to identify donor UTC or MSCs. After 35 days we found E5204positively stained cells in few brain sections of UTC and MSCs treatedgroups. The cells were seen primarily in the lesion boundary zone. Nopositively stained cells were found in control group. (See, FIGS. 25Aand 25B)

BrdU positive cells were seen in UTC (See, FIG. 26A) and MSC (See, FIG.26B) primarily in the lesion boundary zone (See, FIG. 27), indicatingneo-cellular proliferation. Very few cells were visible in the dentategyms. (See, FIG. 28). There was, however, no difference between thetreated and control groups of animals. (See, FIGS. 27 and 28)

vWF/DAB staining was performed to identify angiogenesis in UTC. (See,FIG. 29A) and MSC (See, FIG. 29B). There was no statistical differencein the number of positively stained vessels in treated versus controlgroup of animals in either the boundary zone or dentate gyms. (See, FIG.30 and FIG. 31)

For identification of phenotypes of newly generated cells, sections werestained with Map-2 (neuronal marker) and vWF (endothelial marker).Overlap was found between BrdU and Map-2 in some of the treated groupsof animals. Positive overlap between BrdU and the Map-2immunohistochemistry indicated that newly proliferating cells candifferentiate into neurons (See, FIG. 32). No positive double stainingwas visible in control animals. Cells were also double stained with BrdUand vWF. No overlap was visible between them.

EXAMPLE 11 Trophic Factors for Neural Progenitor Support

The influence of UTC on adult neural stem and progenitor cell survivaland differentiation through non-contact dependent (trophic) mechanismswas examined.

Fisher 344 adult rats were sacrificed by CO₂ asphyxiation followed bycervical dislocation. Whole brains were removed intact using bonerongeurs and hippocampus tissue dissected based on coronal incisionsposterior to the motor and somatosensory regions of the brain (Paxinos,G, & Watson, C, 1997, The Rat Brain in Stereotaxic Coordinates). Tissuewas washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.)containing B27 (B27 supplement; Invitrogen), L-glutamine (4 mM;Invitrogen), and penicillin/streptomycin (Invitrogen), the combinationof which is herein referred to as Neural Progenitor Expansion (NPE)medium. NPE medium was further supplemented with bFGF (20 ng/ml,Peprotech, Rocky Hill, N.J.) and EGF (20 ng/ml, Peprotech, Rocky Hill,N.J.), herein referred to as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 μl per 8 mltotal volume, Sigma, St. Louis, Mo.) was also added. Next, thetissue/media was sequentially passed through an 18 gauge needle, 20gauge needle, and finally a 25 gauge needle one time each (all needlesfrom Becton Dickinson, Franklin Lakes, N.J.). The mixture wascentrifuged for 3 minutes at 250×g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 μm cell strainer (Becton Dickinson),plated on laminin-coated T-75 flasks (Becton Dickinson) or low cluster24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGF media untilsufficient cell numbers were obtained for the studies outlined.

Umbilical cord tissue-derived cells (umbilicus (022803) P12, (042103)P12, (071003) P12) previously grown in growth medium were plated at5,000 cells/transwell insert (sized for 24 well plate) and grown for aperiod of one week in growth medium in inserts to achieve confluence.

Neural progenitors, grown as neurospheres or as single cells, wereseeded onto laminin-coated 24 well plates at an approximate density of2,000 cells/well in NPE+bFGF+EGF for a period of one day to promotecellular attachment. One day later, transwell inserts containing UTCwere added according to the following scheme:

-   -   (1) Transwell (umbilicus-derived cells in growth media, 200        μl)+neural progenitors (NPE+bFGF+EGF, 1 ml).    -   (2) Transwell (adult human dermal fibroblasts [1F1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 μl)+neural        progenitors (NPE+bFGF+EGF, 1 ml).    -   (3) Control: neural progenitors alone (NPE+bFGF+EGF, 1 ml).    -   (4) Control: neural progenitors alone (NPE only, 1 ml).

After 7 days in co-culture, all conditions were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for a period of 10 minutes at room temperature.Immunocytochemistry was performed using antibodies directed against theepitopes listed in Table 11-1. Briefly, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibody solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 μm DAPI (Molecular Probes)applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

TABLE 11-1 Primary Antibodies Used Antibody Concentration Vendor Rat 401(nestin) 1:200 Chemicon, Temecula, Ca. TuJ1 (BIII Tubulin) 1:500 Sigma,St. Louis, MO Tyrosine hydroxylase (TH) 1:1000 Chemicon GABA 1:400Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, Ca. Myelin BasicProtein (MBP) 1:400 Chemicon

Quantification of hippocampal neural progenitor differentiation wasexamined. A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

To identify unique, secreted factors as a result of co-culture,conditioned media samples taken prior to culture fixation were frozendown at −80° C. overnight. Samples were then applied to ultrafiltrationspin devices (MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography columns (anti-Hu-albumin; IgY) (immunoaffinity did notremove albumin from the samples). Filtrate was analyzed by MALDI. Thepass through was applied to Cibachron Blue affinity chromatographycolumns. Samples were analyzed by SDS-PAGE and 2D gel electrophoresis.

Following culture with umbilicus-derived cells, co-cultured neuralprogenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified. Umbilicus-derived cellssignificantly enhanced the number of mature oligodendrocytes (MBP)(24.0% vs 0% in both control conditions). Furthermore, co-cultureenhanced the number of GFAP+ astrocytes and TuJ1+ neurons in culture(47.2% and 8.7% respectively). These results were confirmed by nestinstaining indicating that progenitor status was lost following co-culture(13.4% vs 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did, however,appear to enhance the survival of neural progenitors.

TABLE 11-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond.1] [Cond. 4] [Cond.5] TuJ1  8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

Conditioned media from umbilicus-derived co-cultures, along with theappropriate controls (NPE media ±1.7% serum, media from co-culture withfibroblasts), were examined for differences. Potentially uniquecompounds were identified and excised from their respective 2D gels.

Co-culture of adult neural progenitor cells with umbilicus-derived cellsresults in differentiation of those cells. Results presented in thisexample indicate that the differentiation of adult neural progenitorcells following co-culture with umbilicus-derived cells is particularlyprofound. Specifically, a significant percentage of matureoligodendrocytes was generated in co-cultures of umbilicus-derivedcells. In view of the lack of contact between the umbilicus-derivedcells and the neural progenitors, this result appears to be a functionof soluble factors released from the umbilicus-derived cells (trophiceffect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+ (indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H, et al., Nature, 2002;417:29-32).

EXAMPLE 12 Short-Term Neural Differentiation of Cells

The ability of umbilicus-derived cells to differentiate into neurallineage cells was examined. Umbilical cord tissues were isolated andexpanded as described in Example 12.

A modified Woodbury-Black protocol, which was originally performed totest the neural induction potential of bone marrow stromal cells, wasused to assess the ability of UTC to differentiate into neural lineagecells (Woodbury, D, et al. J Neurosci. Research, 2000; 61(4):364-70).Briefly, UTC (022803) P4 were thawed and culture expanded in growthmedia at 5,000 cells/cm2 until sub-confluence (75%) was reached. Cellswere then trypsinized and seeded at 6,000 cells per well of a TitretekII glass slide (VWR International, Bristol, Conn.). As controls,mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville, Md.),osteoblasts (P5; CC2538; Cambrex), adipose-derived cells (Artecel, U.S.Pat. No. 6,555,374B1) (P6; Donor 2) and neonatal human dermalfibroblasts (P6; CC2509; Cambrex) were also seeded under the sameconditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20ng/ml; Peprotech, Rocky Hill, N.J.), epidermal growth factor (EGF; 20ng/ml; Peprotech) and penicillin/streptomycin (Invitrogen). After fourdays, cells were rinsed in phosphate-buffered saline (PBS; Invitrogen)and were subsequently cultured in DMEM/F12 medium+20% (v/v)FBS+penicillin/streptomycin for 24 hours. After 24 hours, cells wererinsed with PBS. Cells were then cultured for 1-6 hours in an inductionmedium which was comprised of DMEM/F12 (serum-free) containing 200 mMbutylated hydroxyanisole, 10 μM □potassium chloride, 5 mg/ml insulin, 10μM forskolin, 4 μM valproic acid, and 2 μM □hydrocortisone (allchemicals from Sigma, St. Louis, Mo.). Cells were then fixed in 100%ice-cold methanol and immunocytochemistry was performed (see methodsbelow) to assess human nestin protein expression.

UTC (022803 P11) and adult human dermal fibroblasts (1F1853, P11) werethawed and culture expanded in growth medium at 5,000 cells/cm2 untilsub-confluence (75%) was reached. Cells were then trypsinized and seededat similar density as disclosed above, but onto (1) 24 well tissueculture-treated plates (TCP, Falcon brand, VWR International), (2) TCPwells+2% (w/v) gelatin adsorbed for 1 hour at room temperature, or (3)TCP wells+20 μg/milliliter adsorbed mouse laminin (adsorbed for aminimum of 2 hours at 37° C.; Invitrogen).

As disclosed above, cells were initially expanded and media switched atthe aforementioned timeframes. One set of cultures was fixed, as before,at 5 days and six hours, this time with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature. In thesecond set of cultures, medium was removed and switched to NeuralProgenitor Expansion medium (NPE) consisting of Neurobasal-A medium(Invitrogen) containing B27 (B27 supplement; Invitrogen), L-glutamine (4mM), and penicillin/streptomycin (Invitrogen). NPE medium was furthersupplemented with retinoic acid (RA; 1 μM; Sigma). This medium wasremoved 4 days later and cultures were fixed with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature, and stainedfor nestin, GFAP, and TuJ1 protein expression (see Table 12-1).

TABLE 12-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, Ca. Human Nestin 1:100Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, Ca. Tyrosine hydroxylase (TH) 1:1000Chemicon GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemiconalpha-alpha-smooth muscle 1:400 Sigma actin Human nuclear protein (hNuc)1:150 Chemicon

Umbilicus-derived cells (042203; P11) adult human dermal fibroblasts(P11; 1F1853; Cambrex) were thawed and culture expanded in growth mediumat 5,000 cells/cm2 until sub-confluence (75%) was reached. Cells werethen trypsinized and seeded at 2,000 cells/cm2, but onto 24 well platescoated with laminin (BD Biosciences, Franklin Lakes, N.J.) in thepresence of NPE media supplemented with bFGF (20 ng/ml; Peprotech, RockyHill, N.J.) and EGF (20 ng/ml; Peprotech) [whole media compositionfurther referred to as NPE+F+E]. At the same time, adult rat neuralprogenitors isolated from hippocampus (P4; (062603) were also platedonto 24 well laminin-coated plates in NPE+F+E media. All cultures weremaintained in such conditions for a period of 6 days (cells were fedonce during that time) at which time media was switched to thedifferentiation conditions listed for an additional period of 7 days.Cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for10 minutes at room temperature, and stained for human or rat nestin,GFAP, and TuJ1 protein expression.

TABLE 12-2 Summary of Conditions for Two-Stage Differentiation ProtocolCOND. PRE-DIFFEREN- 2^(nd) STAGE # TIATION DIFF 1 NPE + F (20 ng/ml) +NPE + SHH (200 ng/ml) + E (20 ng/ml) F8 (100 ng/ml) 2 NPE + F (20ng/ml) + NPE + SHH (200 ng/ml) + E (20 ng/ml) F8 (100 ng/ml) +RA (1 μM)3 NPE + F (20 ng/ml) + NPE + RA (1 μM) E (20 ng/ml) 4 NPE + F (20ng/ml) + NPE + F (20 ng/ml) + E (20 ng/ml) E (20 ng/ml) 5 NPE + F (20ng/ml) + Growth Medium E (20 ng/ml) 6 NPE + F (20 ng/ml) + Condition1B + E (20 ng/ml) MP52 (20 ng/ml) 7 NPE + F (20 ng/ml) + Condition 1B +E (20 ng/ml) BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + Condition 1B + E (20ng/ml) GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + Condition 2B + E (20ng/ml) MP52 (20 ng/ml) 10 NPE + F (20 ng/ml) + Condition 2B + E (20ng/ml) BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + Condition 2B + E (20ng/ml) GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + Condition 3B + E (20ng/ml) MP52 (20 ng/ml) 13 NPE + F (20 ng/ml) + Condition 3B + E (20ng/ml) BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + Condition 3B + E (20ng/ml) GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + NPE + MP52 (20 ng/ml) E(20 ng/ml) 16 NPE + F (20 ng/ml) + NPE + BMP7 (20 ng/ml) E (20 ng/ml) 17NPE + F (20 ng/ml) + NPE + GDNF (20 ng/ml) E (20 ng/ml)

Umbilicus-derived cells (P11; (042203)) were thawed and culture expandedin growth medium at 5,000 cells/cm2 until sub-confluence (75%) wasreached. Cells were then trypsinized and seeded at 2,000 cells/cm2, onto24 well laminin-coated plates (BD Biosciences) in the presence of NPE+F(20 ng/ml)+E (20 ng/ml). In addition, some wells contained NPE+F+E+2%FBS or 10% FBS. After four days of “pre-differentiation” conditions, allmedia were removed and samples were switched to NPE medium supplementedwith sonic hedgehog (SHH; 200 ng/ml; Sigma, St. Louis, Mo.), FGF8 (100ng/ml; Peprotech), BDNF (40 ng/ml; Sigma), GDNF (20 ng/ml; Sigma), andretinoic acid (1 μM; Sigma). Seven days post medium change, cultureswere fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10minutes at room temperature, and stained for human nestin, GFAP, TuJ1,desmin, and alpha-smooth muscle actin expression.

Adult rat hippocampal progenitors (062603) were plated as neurospheresor single cells (10,000 cells/well) onto laminin-coated 24 well dishes(BD Biosciences) in NPE+F (20 ng/ml)+E (20 ng/ml).

Separately, umbilicus-derived cells (042203) P11 were thawed and cultureexpanded in NPE+F (20 ng/ml)+E (20 ng/ml) at 5,000 cells/cm2 for aperiod of 48 hours. Cells were then trypsinized and seeded at 2,500cells/well onto existing cultures of neural progenitors. At that time,existing medium was exchanged for fresh medium. Four days later,cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for10 minutes at room temperature, and stained for human nuclear protein(hNuc; Chemicon) (Table 11-1 above) to identify UTC.

Immunocytochemistry was performed using the antibodies listed in Table11-1. Cultures were washed with phosphate-buffered saline (PBS) andexposed to a protein blocking solution containing PBS, 4% (v/v) goatserum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Upon incubation in the first neural induction composition listed above,using glass slides, all cell types transformed into cells with bipolarmorphologies and extended processes. Other larger non-bipolarmorphologies were also observed. Furthermore, the induced cellpopulations stained positively for nestin, a marker of multipotentneural stem and progenitor cells.

When repeated on tissue culture plastic (TCP) dishes, as described inthe second neural induction composition listed above, nestin expressionwas not observed unless laminin was pre-adsorbed to the culture surface.To further assess whether nestin-expressing cells could then go on togenerate mature neurons, UTC and fibroblasts were exposed to NPE+RA (1μM), a media composition known to induce the differentiation of neuralstem and progenitor cells into such cells (Jang, Y K, et al., J.Neurosci. Research, 2004; 75(4):573-84; Jones-Villeneuve, E M, et al.,Mol Cel Biol., 1983; 3(12):2271-9; Mayer-Proschel, M, et al., Neuron,1997; 19(4):773-85). Cells were stained for TuJ1, a marker for immatureand mature neurons, GFAP, a marker of astrocytes, and nestin. Under noconditions was TuJ1 detected, nor were cells with neuronal morphologyobserved, suggesting that neurons were not generated in the short term.Furthermore, nestin and GFAP were no longer expressed by UTC, asdetermined by immunocytochemistry.

UTC isolates (as well as human fibroblasts and rodent neural progenitorsas negative and positive control cell types, respectively) were platedon laminin (neural promoting)-coated dishes and exposed to 13 differentgrowth conditions (and two control conditions) known to promotedifferentiation of neural progenitors into neurons and astrocytes. Inaddition, two conditions were added to examine the influence of GDF5,and BMP7 on PPDC differentiation. Generally, a two-step differentiationapproach was taken, where the cells were first placed in neuralprogenitor expansion conditions for a period of 6 days, followed by fulldifferentiation conditions for 7 days. Morphologically, both umbilicus-and placenta-derived cells exhibited fundamental changes in cellmorphology throughout the time-course of this procedure. However,neuronal or astrocytic-shaped cells were not observed except for incontrol, neural progenitor-plated conditions. Immunocytochemistry,negative for human nestin, TuJ1, and GFAP confirmed the morphologicalobservations.

Following one week's exposure to a variety of neural differentiationagents, cells were stained for markers indicative of neural progenitors(human nestin), neurons (TuJ1), and astrocytes (GFAP). Cells grown inthe first stage in non-serum containing media had different morphologiesthan those cells in serum containing (2% or 10%) media, indicatingpotential neural differentiation. Specifically, following a two stepprocedure of exposing umbilicus-derived cells to EGF and bFGF, followedby SHH, FGF8, GDNF, BDNF, and retinoic acid, cells showed long extendedprocesses similar to the morphology of cultured astrocytes. When 2% FBSor 10% FBS was included in the first stage of differentiation, cellnumber was increased and cell morphology was unchanged from controlcultures at high density. Potential neural differentiation was notevidenced by immunocytochemical analysis for human nestin, TuJ1, orGFAP.

UTC were plated onto cultures of rat neural progenitors seeded two daysearlier in neural expansion conditions (NPE+F+E). While visualconfirmation of plated UTC proved that these cells were plated as singlecells, human-specific nuclear staining (hNuc) 4 days post-plating (6days total) showed that they tended to ball up and avoid contact withthe neural progenitors. Furthermore, where UTC attached, these cellsspread out and appeared to be innervated by differentiated neurons thatwere of rat origin, suggesting that the UTC may have differentiated intomuscle cells. This observation was based upon morphology under phasecontrast microscopy. Another observation was that typically large cellbodies (larger than neural progenitors) possessed morphologiesresembling neural progenitors, with thin processes spanning out inmultiple directions. HNuc staining (found in one half of the cell'snucleus) suggested that in some cases these human cells may have fusedwith rat progenitors and assumed their phenotype. Control wellscontaining only neural progenitors had fewer total progenitors andapparent differentiated cells than did co-culture wells containingumbilicus or placenta-derived cells, further indicating that bothumbilicus- and placenta-derived cells influenced the differentiation andbehavior of neural progenitors, either by release of chemokines andcytokines, or by contact-mediated effects.

Multiple protocols were conducted to determine the short term potentialof UTC to differentiate into neural lineage cells. These included phasecontrast imaging of morphology in combination with immunocytochemistryfor nestin, TuJ1, and GFAP, proteins associated with multipotent neuralstem and progenitor cells, immature and mature neurons, and astrocytes,respectively. Evidence was observed to suggest that neuraldifferentiation occurred in certain instances in these short-termprotocols.

Several notable observations were made in co-cultures of UTC with neuralprogenitors. This approach, using human UTC along with a xenogeneic celltype allowed for absolute determination of the origin of each cell inthese cultures. First, some cells were observed in these cultures wherethe cell cytoplasm was enlarged, with neurite-like processes extendingaway from the cell body, yet only half of the body labeled with hNucprotein. Those cells may have been human UTC that had differentiatedinto neural lineage cells or they may have been UTC that had fused withneural progenitors. Second, it appeared that neural progenitors extendedneurites to UTC in a way that indicates the progenitors differentiatedinto neurons and innervated the UTC. Third, cultures of neuralprogenitors and UTC had more cells of rat origin and larger amounts ofdifferentiation than control cultures of neural progenitors alone,further indicating that plated UTC provided soluble factors and orcontact-dependent mechanisms that stimulated neural progenitor survival,proliferation, and/or differentiation.

EXAMPLE 13 Isolation of Cells

Umbilical cords were obtained from National Disease Research Interchange(NDRI, Philadelphia, Pa.). The tissues were obtained following normaldeliveries. The cell isolation protocols were performed aseptically in alaminar flow hood. To remove blood and debris, the cord was washed inphosphate buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in thepresence of penicillin at 100 Units/milliliter, streptomycin at 100milligrams/milliliter and amphotericin B at 0.025 micrograms/milliliter(Invitrogen Carlsbad, Calif.). The tissues were then mechanicallydissociated in 150 cm² tissue culture plates in the presence of 50milliliters of medium (DMEM-low glucose or DMEM-high glucose;Invitrogen), until the tissue was minced into a fine pulp. The choppedtissues were transferred to 50 milliliter conical tubes (approximately 5grams of tissue per tube).

The tissue was then digested in either DMEM-low glucose medium orDMEM-high glucose medium, each containing penicillin at 100Units/milliliter, streptomycin at 100 milligrams/milliliter,amphotericin B at 0.25 micrograms/milliliter and the digestion enzymes.In some experiments an enzyme mixture of collagenase and dispase wasused (“C:D”) (collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter;and dispase (Invitrogen), 50 Units/milliliter, in DMEM-Low glucosemedium). In other experiments a mixture of collagenase, dispase andhyaluronidase (“C:D:H”) was used (C:D:H=collagenase, 500Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase(Sigma), 5 Units/milliliter, in DMEM-low glucose). The conical tubescontaining the tissue, medium and digestion enzymes were incubated at37° C. in an orbital shaker (Environ, Brooklyn, N.Y.) at 225 rpm for 2hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the supernatant was aspirated. The pellet was resuspended in 20milliliters of growth medium (DMEM: low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined fetal bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol(Sigma), penicillin at 100 Units per milliliter, streptomycin at 100micrograms per milliliter, and amphotericin B at 0.25 micrograms permilliliter; (each from Invitrogen, Carlsbad, Calif.)). The cellsuspension was filtered through a 70-micron nylon BD FALCON CellStrainer (BD Biosciences, San Jose, Calif.). An additional 5 millilitersrinse comprising growth medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences, San Jose, Calif.) and chased with a rinse of anadditional 5 milliliters of growth medium.

The filtrate was resuspended in growth medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshgrowth medium. This process was repeated twice more.

After the final centrifugation, supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh growth medium. Thenumber of viable cells was determined using trypan blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cord tissues were seeded at 5,000cells/cm² onto gelatin-coated T-75 flasks (Corning Inc., Corning, N.Y.)in growth medium. After two days, spent medium and unadhered cells wereaspirated from the flasks. Adherent cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with growth medium and allowed to grow to confluence (about10 days from passage 0 to passage 1). On subsequent passages (frompassage 1 to 2 etc), cells reached sub-confluence (75-85 percentconfluence) in 4-5 days. For these subsequent passages, cells wereseeded at 5,000 cells/cm². Cells were grown in a humidified incubatorwith 5 percent carbon dioxide at 37° C.

In some experiments, cells were isolated from umbilical cord tissues inDMEM-low glucose medium after digestion with LIBERASE (2.5 milligramsper milliliter, BLENDZYME 3; Roche Applied Sciences, Indianapolis, Ind.)and hyaluronidase (5 Units/milliliter, Sigma). Digestion of the tissueand isolation of the cells was as described for other proteasedigestions above, however, the LIBERASE/hyaluronidase mixture was usedinstead of the C:D or C:D:H enzyme mixture. Tissue digestion withLIBERASE resulted in the isolation of cell populations from postpartumtissues that expanded readily.

Procedures were compared for isolating cells from the umbilical cordusing differing enzyme combinations. Enzymes compared for digestionincluded: i) collagenase; ii) dispase; iii) hyaluronidase; iv)collagenase:dispase mixture (C:D); v) collagenase:hyaluronidase mixture(C:H); vi) dispase:hyaluronidase mixture (D:H); and vii)collagenase:dispase:hyaluronidase mixture (C:D:H). Differences in cellisolation utilizing these different enzyme digestion conditions wereobserved (Table 13-1).

Other attempts were made to isolate pools of cells from umbilical cordby different approaches. In one instance, umbilical cord was sliced andwashed with growth medium to dislodge the blood clots and gelatinousmaterial. The mixture of blood, gelatinous material and growth mediumwas collected and centrifuged at 150×g. The pellet was resuspended andseeded onto gelatin coated flasks in growth medium. From theseexperiments a cell population was isolated that readily expanded.

Cells have also been isolated from cord blood samples obtained fromNDRI. The isolation protocol used was that of International PatentApplication PCT/US2002/029971 by Ho et al. Samples (50 milliliter and10.5 milliliters, respectively) of umbilical cord blood (NDRI,Philadelphia Pa.) were mixed with lysis buffer (filter-sterilized 155millimolar ammonium chloride, 10 millimolar potassium bicarbonate, 0.1millimolar EDTA buffered to pH 7.2 (all components from Sigma, St.Louis, Mo.). Cells were lysed at a ratio of 1:20 cord blood to lysisbuffer. The resulting cell suspension was vortexed for 5 seconds, andincubated for 2 minutes at ambient temperature. The lysate wascentrifuged (10 minutes at 200×g). The cell pellet was resuspended inComplete Minimal Essential Medium (Gibco, Carlsbad Calif.) containing 10percent fetal bovine serum (Hyclone, Logan Utah), 4 millimolar glutamine(Mediatech Herndon, Va.), penicillin at 100 Units per milliliter andstreptomycin at 100 micrograms per milliliter (Gibco, Carlsbad, Calif.).The resuspended cells were centrifuged (10 minutes at 200×g), thesupernatant was aspirated, and the cell pellet was washed in completemedium. Cells were seeded directly into either T75 flasks (Corning,N.Y.), T75 laminin-coated flasks, or T175 fibronectin-coated flasks(both Becton Dickinson, Bedford, Mass.).

To determine whether cell populations could be isolated under differentconditions and expanded under a variety of conditions immediately afterisolation, cells were digested in growth medium with or without 0.001percent (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using theenzyme combination of C:D:H, according to the procedures provided above.All cells were grown in the presence of penicillin at 100 Units permilliliter and streptomycin at 100 micrograms per milliliter. Under alltested conditions cells attached and expanded well between passage 0 and1 (Table 13-2). Cells in conditions 5-8 and 13-16 were demonstrated toproliferate well up to 4 passages after seeding, at which point theywere cryopreserved.

The combination of C:D:H, provided the best cell yield followingisolation, and generated cells that expanded for many more generationsin culture than the other conditions (Table 13-1). An expandable cellpopulation was not attained using collagenase or hyaluronidase alone. Noattempt was made to determine if this result is specific to thecollagenase that was tested.

TABLE 13-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Enzyme Digest Cells Isolated Cell ExpansionCollagenase X X Dispase     + (>10 h) + Hyaluronidase X XCollagenase:Dispase   ++ (<3 h)  ++ Collagenase:Hyaluronidase   ++ (<3h)  + Dispase:Hyaluronidase     + (>10 h) +Collagenase:Dispase:Hyaluronidase +++ (<3 h)  +++ Key: + = good, ++ =very good, +++ = excellent, X = no success under conditions tested

Cells attached and expanded well between passage 0 and 1 under allconditions tested for enzyme digestion and growth. Cells in experimentalconditions 5-8 and 13-16 proliferated well up to 4 passages afterseeding, at which point they were cryopreserved. All cells werecryopreserved for further analysis.

TABLE 13-2 Isolation and culture expansion of umbilical cord cells undervarying conditions: Condition Medium 15% FBS BME Gelatin 20% O₂ GrowthFactors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7DMEM-Lg N (2%) Y N (Fibrone) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N (Fibrone)N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N(Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N (5%)EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N (Fibrone) Y PDGF/VEGF 16DMEM-Lg N (2%) N N (Fibrone) N (5%) PDGF/VEGF

Nucleated cells attached and grew rapidly. These cells were analyzed byflow cytometry and were similar to cells obtained by enzyme digestion.

The preparations contained red blood cells and platelets. No nucleatedcells attached and divided during the first 3 weeks. The medium waschanged 3 weeks after seeding and no cells were observed to attach andgrow.

Populations of cells could be isolated from umbilical tissue efficientlyusing the enzyme combination collagenase (a metalloprotease), dispase(neutral protease) and hyaluronidase (mucolytic enzyme which breaks downhyaluronic acid). LIBERASE, which is a blend of collagenase and aneutral protease, may also be used. BLENDZYME 3, which is collagenase (4Wunsch units/gram) and thermolysin (1714 casein Units/gram), was alsoused together with hyaluronidase to isolate cells. These cells expandedreadily over many passages when cultured in growth expansion medium ongelatin coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue,which adhere and grow under the conditions used, may be due to cellsbeing released during the dissection process.

EXAMPLE 14 Growth Characteristics of Cells

The cell expansion potential of umbilical cord tissue-derived cells wascompared to other populations of isolated stem cells. The process ofcell expansion to senescence is referred to as Hayflick's limit(Hayflick, L, J. Am. Geriatr. Soc., 1974; 22(1):1-12; Hayflick, L,Gerontologist, 1974; 14(1):37-45), 1974).

Tissue culture plastic flasks were coated by adding 20 milliliters 2%(w/v) gelatin (Type B: 225 Bloom; Sigma, St Louis, Mo.) to a T75 flask(Corning Inc., Corning, N.Y.) for 20 minutes at room temperature. Afterremoving the gelatin solution, 10 milliliters of phosphate-bufferedsaline (PBS) (Invitrogen, Carlsbad, Calif.) was added and thenaspirated.

For comparison of growth expansion potential the following cellpopulations were utilized; i) mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) adipose-derived cells (U.S. Pat. No. 6,555,374B1; U.S. Patent Application US20040058412); iii) normal dermal skinfibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.); and iv)umbilicus-derived cells. Cells were initially seeded at 5,000 cells/cm²on gelatin-coated T75 flasks in growth medium. For subsequent passages,cell cultures were treated as follows. After trypsinization, viablecells were counted after trypan blue staining. Cell suspension (50microliters) was combined with trypan blue (50 microliters, Sigma, St.Louis Mo.). Viable cell numbers were estimated using a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh growth medium.Cells were grown in a standard atmosphere (5 percent carbon dioxide(v/v)) at 37° C. The growth medium was changed twice per week. Whencells reached about 85 percent confluence they were passaged; thisprocess was repeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doublings [ln (cells final/cells initial)/ln 2], anddoubling time (time in culture/population doubling) were calculated. Forthe purposes of determining optimal cell expansion, the total cell yieldper passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cells final/cells initial).

The expansion potential of cells banked at passage 10 was also tested. Adifferent set of conditions was used. Normal dermal skin fibroblasts(cc-2509 lot #9F0844; Cambrex, Walkersville, Md.), umbilicus-derivedcells were tested. These cell populations had been banked at passage 10previously, having been cultured at 5,000 cells/cm² at each passage tothat point. The effect of cell density on the cell populations followingcell thaw at passage 10 was determined. Cells were thawed under standardconditions, and counted using trypan blue staining. Thawed cells werethen seeded at 1,000 cells/cm² in growth medium. Cells were grown understandard atmospheric conditions at 37° C. growth medium was changedtwice a week. Cells were passaged as they reached about 85% confluence.Cells were subsequently passaged until senescence, i.e., until theycould not be expanded any further. Cells were trypsinized and counted ateach passage. The cell yield, population doubling (ln (cells final/cellsinitial)/ln 2) and doubling time (time in culture/population doubling)were calculated. The total cell yield per passage was determined bymultiplying total yield for the previous passage by the expansion factorfor each passage (i.e., expansion factor=cells final/cells initial).

The expansion potential of freshly isolated umbilical cordtissue-derived cell cultures under low cell seeding conditions wastested in another experiment. Umbilicus-derived cells were isolated asdescribed in Example 12. Cells were seeded at 1,000 cells/cm² andpassaged as described above until senescence. Cells were grown understandard atmospheric conditions at 37° C. Growth medium was changedtwice per week. Cells were passaged as they reached about 85%confluence. At each passage, cells were trypsinized and counted bytrypan blue staining. The cell yield, population doubling (ln (cellfinal/cell initial)/ln 2) and doubling time (time in culture/populationdoubling) were calculated for each passage. The total cell yield perpassage was determined by multiplying the total yield for the previouspassage by the expansion factor for each passage (i.e., expansionfactor=cell final/cell initial). Cells were grown on gelatin andnon-gelatin coated flasks.

It has been demonstrated that low O₂ cell culture conditions can improvecell expansion in certain circumstances (United States PatentApplication No. US20040005704). To determine if cell expansion of UTCcould be improved by altering cell culture conditions, cultures ofumbilicus-derived cells were grown in low oxygen conditions. Cells wereseeded at 5,000 cells/cm² in growth medium on gelatin coated flasks.Cells were initially cultured under standard atmospheric conditionsthrough passage 5, at which point they were transferred to low oxygen(5% O₂) culture conditions.

In other experiments cells were expanded on non-coated, collagen-coated,fibronectin-coated, laminin-coated and matrigel-coated plates. Cultureshave been demonstrated to expand well on these different matrices.

Umbilicus-derived cells expanded for more than 40 passages generatingcell yields of >1×10¹⁷ cells in 60 days. In contrast, MSCs andfibroblasts senesced after <25 days and <60 days, respectively. Althoughboth adipose-derived and omental cells expanded for almost 60 days, theygenerated total cell yields of 4.5×10¹² and 4.24×10¹³ respectively.Thus, when seeded at 5,000 cells/cm² under the experimental conditionsutilized, umbilicus-derived cells expanded much better than the othercell types grown under the same conditions (Table 14-1).

TABLE 14-1 Growth characteristics for different cell populations grownto senescence Total Population Total Cell Cell Type Senescence DoublingsYield MSC 24 days 8 4.72 × 10⁷  Adipose 57 days 24  4.5 × 10¹²Fibroblasts 53 days 26 2.82 × 10¹³ Umbilicus 65 days 42 6.15 × 10¹⁷

Umbilicus-derived cells and fibroblast cells expanded for greater than10 passages generating cell yields of >1×10¹¹ cells in 60 days (Table14-2). After 60 days under these conditions the fibroblasts becamesenescent whereas the umbilicus-derived cell populations senesced after80 days, completing >40 population doublings.

TABLE 14- 2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 till senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 days 43.68 2.59 × 10¹¹ Umbilicus (P10) 80 days 53.6 1.25 × 10¹⁴

Cells expanded well under the reduced oxygen conditions, however,culturing under low oxygen conditions does not appear to have asignificant effect on cell expansion for umbilical cord tissue-derivedcells. Standard atmospheric conditions have already proven successfulfor growing sufficient numbers of cells, and low oxygen culture is notrequired for the growth of umbilical cord tissue-derived cells.

The current cell expansion conditions of growing isolated umbilical cordtissue-derived cells at densities of about 5,000 cells/cm², in growthmedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1,000 cells/cm²). Umbilicalcord tissue derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing umbilical cordtissue-derived cells under standard atmospheric conditions is preferredfor generating large pools of cells. When the culture conditions arealtered, however, umbilical cord tissue-derived cell expansion canlikewise be altered. This strategy may be used to enhance theproliferative and differentiative capacity of these cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, UTC expand readily to large numbers.

EXAMPLE 15 Growth of Cells in Medium Containing D-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan J. Cell Biol Int., 2000;24:1-7; Sordillo L M, et al., Cell Biol Int Rep., 1988; 12:355-64).Experiments were performed to determine whether umbilical cordtissue-derived cells could grow in medium containing D-valine.

Umbilicus-derived cells (P5) and fibroblasts (P9) were seeded at 5,000cells/cm² in gelatin-coated T75 flasks (Corning, Corning, N.Y.). After24 hours the medium was removed and the cells were washed with phosphatebuffered saline (PBS) (Gibco, Carlsbad, Calif.) to remove residualmedium. The medium was replaced with a modified growth medium (DMEM withD-valine (special order Gibco), 15% (v/v) dialyzed fetal bovine serum(Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma),penicillin at 50 Units/milliliter and streptomycin at 50milligrams/milliliter (Gibco)).

Umbilicus-derived cells and fibroblast cells seeded in theD-valine-containing medium did not proliferate, unlike cells seeded ingrowth medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after four weeks.Thus, it may be concluded that umbilical cord tissue-derived cellsrequire L-valine for cell growth and to maintain long-term viability.L-valine is preferably not removed from the growth medium for umbilicalcord tissue-derived cells.

EXAMPLE 16 Karyotype Analysis of Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Human cells used in cell therapy shouldhave a normal number (46) of chromosomes with normal structure. Toidentify umbilical cord tissue-derived cell lines that are homogeneousand free from cells of non-postpartum tissue origin, karyotypes of cellsamples were analyzed.

Umbilical cord tissue-derived cells from postpartum tissue of a maleneonate were cultured in growth media. Umbilical cord tissue from a maleneonate (X,Y) was selected to allow distinction between neonatal-derivedcells and maternal derived cells (X,X). Cells were seeded at 5,000 cellsper square centimeter in growth medium in a T25 flask (Corning, Corning,N.Y.) and expanded to 80% confluence. A T25 flask containing cells wasfilled to the neck with growth media. Samples were delivered to aclinical cytogenetics lab by courier (estimated lab to lab transporttime is one hour). Chromosome analysis was performed by the Center forHuman & Molecular Genetics at the New Jersey Medical School, Newark,N.J. Cells were analyzed during metaphase when the chromosomes are bestvisualized. Of twenty cells in metaphase counted, five were analyzed fornormal homogeneous karyotype number (two). A cell sample wascharacterized as homogeneous if two karyotypes were observed. A cellsample was characterized as heterogeneous if more than two karyotypeswere observed. Additional metaphase cells were counted and analyzed whena heterogeneous karyotype number (four) was identified.

All cell samples sent for chromosome analysis were interpreted by thecytogenetics laboratory staff as exhibiting a normal appearance. Threeof the sixteen cell lines analyzed exhibited a heterogeneous phenotype(XX and XY) indicating the presence of cells derived from both neonataland maternal origins. Each of the cell samples was characterized ashomogeneous. (Table 16-1).

TABLE 16-1 Results of UTC karyotype analysis Metaphase Metaphase Numbercells cells of ISCN Tissue Passage counted analyzed karyotypes KaryotypeUmbilical 23 20 5 2 46, XX Umbilical 6 20 5 2 46, XY Umbilical 3 20 5 246, XX Key: N-Neonatal aspect; V-villous region; M-maternal aspect;C-clone

Chromosome analysis identified umbilicus-derived cells whose karyotypesappear normal as interpreted by a clinical cytogenetic laboratory.Karyotype analysis also identified cell lines free from maternal cells,as determined by homogeneous karyotype.

EXAMPLE 17 Flow Cytometric Evaluation of Cell Surface Markers

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Cell lines isolatedfrom umbilicus were characterized (by flow cytometry), providing aprofile for the identification of these cell lines.

Cells were cultured in growth medium (Gibco Carlsbad, Calif.) withpenicillin/streptomycin. Cells were cultured in plasma-treated T75,T150, and T225 tissue culture flasks (Corning, Corning, N.Y.) untilconfluent. The growth surfaces of the flasks were coated with gelatin byincubating 2% (w/v) gelatin (Sigma, St. Louis, Mo.) for 20 minutes atroom temperature.

Adherent cells in flasks were washed in PBS and detached withTrypsin/EDTA. Cells were harvested, centrifuged, and resuspended in 3%(v/v) FBS in PBS at a cell concentration of 1×10⁷ per ml. In accordanceto the manufacture's specifications, antibody to the cell surface markerof interest (see below) was added to 100 μl of cell suspension and themixture was incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged to remove unboundantibody. Cells were resuspended in 500 μl PBS and analyzed by flowcytometry. Flow cytometry analysis was performed with a FACS caliburinstrument (Becton Dickinson, San Jose, Calif.).

The following antibodies directed against cell surface markers wereused.

Antibody Manufacture Catalog Number CD10 BD Pharmingen (San Diego, Ca.)555375 CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446 CD34 BDPharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen 555489CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BD Pharmingen340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen 556002HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen 555558IgG-FITC Sigma (St. Louis, Mo.) F-6522 IgG- PE Sigma P-4685

Umbilical cord cells were analyzed at passages 8, 15, and 20.

To compare differences among donors, umbilical cord-derived cells fromdifferent donors were compared to each other.

Umbilical cord-derived cells cultured on gelatin-coated flasks werecompared to umbilical cord-derived cells cultured on uncoated flasks.

Four treatments used for isolation and preparation of cells werecompared. Cells derived from postpartum tissue by treatment with: 1)collagenase; 2) collagenase/dispase; 3) collagenase/hyaluronidase; and4) collagenase/hyaluronidase/dispase were compared.

Umbilical cord-derived cells at passage 8, 15, and 20 analyzed by flowcytometry all expressed CD10, CD13, CD44, CD73, CD90, PDGFr-alpha andHLA-A, B, C, indicated by increased fluorescence relative to the IgGcontrol. These cells were negative for CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ, indicated by fluorescence values consistent with theIgG control.

Umbilical cord-derived cells isolated from separate donors analyzed byflow cytometry each showed positive for production of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for production of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ with fluorescence values consistent with the IgG control.

Umbilical cord-derived cells expanded on gelatin-coated and uncoatedflasks analyzed by flow cytometry were all positive for production ofCD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, withincreased values of fluorescence relative to the IgG control. Thesecells were negative for production of CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ, with fluorescence values consistent with the IgGcontrol.

Analysis of umbilical cord-derived cells by flow cytometry hasestablished an identity of these cell lines. These umbilicalcord-derived postpartum cells are positive for CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, and HLA-A, B, C; and negative for CD31, CD34, CD45,CD117, CD141 and HLA-DR, DP, DQ. This identity was consistent betweenvariations in variables including the donor, passage, culture vesselsurface coating, digestion enzymes, and placental layer. Some variationin individual fluorescence value histogram curve means and ranges wereobserved, but all positive curves under all conditions tested werenormal and expressed fluorescence values greater than the IgG control,thus confirming that the cells comprise a homogeneous population whichhas positive expression of the markers.

EXAMPLE 18 Analysis of Cells by Oligonucleotide Array

Oligonucleotide arrays were used to compare gene expression profiles ofumbilicus-derived and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Human umbilical cords and placenta were obtained from National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.) from normal full termdeliveries with patient consent. The tissues were received and cellswere isolated as described in Example 13 after digestion with a C:D:Hmixture. Cells were cultured in growth medium on gelatin-coated plastictissue culture flasks. The cultures were incubated at 37° C. with 5%CO₂.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Bothlines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.)with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin(Invitrogen)). The cells were grown on standard tissue-treated plastic.

hMSCs were purchased from Cambrex Incorporated (Walkersville, Md.; Lotnumbers 2F1655, 2F1656 and 2F1657) and cultured according to themanufacturer's specifications in MSCGM Media (Cambrex). The cells weregrown on standard tissue cultured plastic at 37° C. with 5% CO₂.

Human iliac crest bone marrow was received from NDRI with patientconsent. The marrow was processed according to the method outlined byHo, et al. (WO03/025149). The marrow was mixed with lysis buffer (155 mMNH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bonemarrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 mM glutamine. The cells werecentrifuged again and the cell pellet was resuspended in fresh medium.The viable mononuclear cells were counted using trypan blue exclusion(Sigma, St. Louis, Mo.). The mononuclear cells were seeded in plastictissue culture flasks at 5×10⁴ cells/cm². The cells were incubated at37° C. with 5% CO₂ at either standard atmospheric O₂ or at 5% O₂. Cellswere cultured for 5 days without a media change. Media and non-adherentcells were removed after 5 days of culture. The adherent cells weremaintained in culture.

Actively growing cultures of cells were removed from the flasks with acell scraper in cold phosphate buffered saline (PBS). The cells werecentrifuged for 5 minutes at 300×g. The supernatant was removed and thecells were resuspended in fresh PBS and centrifuged again. Thesupernatant was removed and the cell pellet was immediately frozen andstored at −80° C. Cellular mRNA was extracted and transcribed into cDNA.cDNA was then transcribed into cRNA and biotin-labeled. Thebiotin-labeled cRNA was hybridized with Affymetrix GENECHIP HG-U133Aoligonucleotide arrays (Affymetrix, Santa Clara, Calif.). Thehybridizations and data collection were performed according to themanufacturer's specifications. The hybridization and data collection wasperformed according to the manufacturer's specifications. Data analyseswere performed using “Significance Analysis of Microarrays” (SAM)version 1.21 computer software (Tusher, V G, et al., Proc. Natl. Acad.Sci. USA, 2001; 98:5116-5121).

Fourteen different populations of cells were analyzed in this study. Thecells along with passage information, culture substrate, and culturemedia are listed in Table 18-1.

TABLE 18-1 Cells analyzed by the microarray study. Cell lines are listedby identification code along with passage at time of analysis, cellgrowth substrate and growth medium. Cell Population Passage SubstrateMedium Umbilicus (022803) 2 Gelatin DMEM, 15% FBS, 2B-ME Umbilicus(042103) 3 Gelatin DMEM, 15% FBS, 2B-ME Umbilicus (071003) 4 GelatinDMEM, 15% FBS, 2B-ME Placenta (042203) 12 Gelatin DMEM, 15% FBS, 2B-MEPlacenta (042903) 4 Gelatin DMEM, 15% FBS, 2B-ME Placenta (071003) 3Gelatin DMEM, 15% FBS, 2B-ME ICBM (070203) (5% O₂) 3 Plastic MEM, 10%FBS ICBM (062703) (std. O₂) 5 Plastic MEM, 10% FBS ICBM (062703) (5% O₂)5 Plastic MEM, 10% FBS hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot2F1656) 3 Plastic MSCGM hMSC (Lot 2F 1657) 3 Plastic MSCGM hFibroblast(9F0844) 9 Plastic DMEM-F12, 10% FBS hFibroblast (CCD39SK) 4 PlasticDMEM-F12, 10% FBS

The data were evaluated by principle component analysis with SAMsoftware as described above. Analysis revealed 290 genes that wereexpressed in different relative amounts in the cells tested. Thisanalysis provided relative comparisons between the populations.

Table 18-2 shows the Euclidean distances that were calculated for thecomparison of the cell pairs. The Euclidean distances were based on thecomparison of the cells based on the 290 genes that were differentiallyexpressed among the cell types. The Euclidean distance is inverselyproportional to similarity between the expression of the 290 genes.

TABLE 18-2 The Euclidean Distances for the Cell Pairs. The Euclideandistance was calculated for the cell types using the 290 genes that wereexpressed differentially between the cell types. Similarity between thecells is inversely proportional to the Euclidean distance. Cell PairEuclidean Distance ICBM-HMSC 24.71 PLACENTA-UMBILICAL 25.52ICBM-FIBROBLAST 36.44 ICBM-PLACENTA 37.09 FIBROBLAST-MSC 39.63ICBM-UMBILICAL 40.15 Fibroblast-Umbilical 41.59 MSC-PLACENTA 42.84MSC-UMBILICAL 46.86 ICBM-PLACENTA 48.41

Tables 18-3, 18-4, and 18-5 show the expression of genes increased inumbilical tissue-derived cells (Table 18-3), increased inplacenta-derived cells (Table 18-4), and reduced in umbilicus- andplacenta-derived cells (Table 18-5).

TABLE 18-3 Genes shown to have specifically increased expression in theUTC as compared to other cell lines assayed Probe Set NCBI Accession IDGene Name Number 202859_x_at interleukin 8 NM_000584 211506_s_atinterleukin 8 AF043337 210222_s_at reticulon 1 BC000314 204470_atchemokine (C-X-C motif) ligand 1 NM_001511 (melanoma growth stimulatingactivity 206336_at chemokine (C-X-C motif) ligand 6 NM_002993(granulocyte chemotactic protein 2) 207850_at chemokine (C-X-C motif)ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumornecrosis factor, alpha-induced NM_006290 protein 3

TABLE 18-4 Genes shown to have specifically increased expression in theplacenta-derived cells as compared to other cell lines assayed Probe SetNCBI Accession ID Gene Name Number 209732_at C-type (calcium dependent,carbohydrate-recognition AF070642 domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 family, member A2 AB015228 206367_at reninNM_000537 210004_at oxidized low density lipoprotein (lectin-like)receptor 1 AF035776 214993_at Homo sapiens, clone IMAGE:4179671, mRNA,partial cds AF070642 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;cDNA DKFZp547K1113 AI246730 (from clone DKFZp547K1113)

TABLE 18-5 Genes shown to have decreased expression in umbilicus-derivedand placenta-derived cells as compared to other cell lines assayed ProbeSet NCBI Accession ID Gene name Number 210135_s_at short staturehomeobox 2 AF022654.1 205824_at heat shock 27kDa protein 2 NM_001541.1209687_at chemokine (C-X-C motif) ligand 12 U19495.1 (stromalcell-derived factor 1) 203666_at chemokine (C-X-C motif) ligand 12NM_000609.1 (stromal cell-derived factor 1) 212670_at elastin(supravalvular aortic stenosis, AA479278 Williams-Beuren syndrome)213381_at Homo sapiens mRNA; cDNA DKFZp586M2022 N91149 (from cloneDKFZp586M2022) 206201_s_at mesenchyme homeo box 2 (growth NM_005924.1arrest-specific homeo box) 205817_at sine oculis homeobox homolog 1(Drosophila) NM_005982.1 209283_at crystallin, alpha B AF007162.1212793_at dishevelled associated activator of morphogenesis 2 BF513244213488_at DKFZP586B2420 protein AL050143.1 209763_at similar to neuralin1 AL049176 205200_at tetranectin (plasminogen binding protein)NM_003278.1 205743_at src homology three (SH3) and cysteine rich domainNM_003149.1 200921_s_at B-cell translocation gene 1, anti-proliferativeNM_001731.1 206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_atrunt-related transcription factor 3 AA541630 219747_at hypotheticalprotein FLJ23191 NM_024574.1 204773_at interleukin 11 receptor, alphaNM_004512.1 202465_at procollagen C-endopeptidase enhancer NM_002593.2203706_s_at frizzled homolog 7 (Drosophila) NM_003507.1 212736_athypothetical gene BC008967 BE299456 214587_at collagen, type VIII, alpha1 BE877796 201645_at tenascin C (hexabrachion) NM_002160.1 210239_atiroquois homeobox protein 5 U90304.1 203903_s_at hephaestin NM_014799.1205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicleglycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis,clone AU147799 MAMMA1001744 206315_at cytokine receptor-like factor 1NM_004750.1 204401_at potassium intermediate/small conductance calcium-NM_002250.1 activated channel, subfamily N, member 4 216331_at integrin,alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1 213125_atDKFZP586L151 protein AW007573 202133_at transcriptional co-activatorwith PDZ-binding motif (TAZ) AA081084 206511_s_at sine oculis homeoboxhomolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034 protein AB028957.1206115_at early growth response 3 NM_004430.1 213707_s_at distal-lesshomeo box 5 NM_005221.3 218181_s_at hypothetical protein FLJ20373NM_017792.1 209160_at aldo-keto reductase family 1, member C3 (3-alphaAB018580.1 hydroxysteroid dehydrogenase, type II) 213905_x_at biglycanAA845258 201261_x_at biglycan BC002416.1 202132_at transcriptionalco-activator with PDZ-binding motif (TAZ) AA081084 214701_s_atfibronectin 1 AJ276395.1 213791_at proenkephalin NM_006211.1 205422_s_atintegrin, beta-like 1 (with EGF-like repeat domains) NM_004791.1214927_at Homo sapiens mRNA full length insert cDNA clone AL359052.1EUROIMAGE 1968422 206070_s_at EphA3 AF213459.1 212805_at KIAA0367protein AB002365.1 219789_at natriuretic peptide receptor C/guanylatecyclase C AI628360 (atrionatriuretic peptide receptor C) 219054_athypothetical protein FLJ14054 NM_024563.1 213429_at Homo sapiens mRNA;cDNA DKFZp564B222 (from AW025579 clone DKFZp564B222) 204929_s_atvesicle-associated membrane protein 5 (myobrevin) NM_006634.1201843_s_at EGF-containing fibulin-like extracellular matrix protein 1NM_004105.2 221478_at BCL2/adenovirus E1B l9kDa interacting protein3-like AL132665.1 201792_at AE binding protein 1 NM_001129.2 204570_atcytochrome c oxidase subunit VIIa polypeptide 1 (muscle) NM_001864.1201621_at neuroblastoma, suppression of tumorigenicity 1 NM_005380.1202718_at insulin-like growth factor binding protein 2, 36kDaNM_000597.1

Tables 18-6, 18-7, and 18-8 show the expression of genes increased inhuman fibroblasts (Table 18-6), ICBM cells (Table 18-7), and MSCs (Table18-8).

TABLE 18-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. dualspecificity phosphatase 2 KIAA0527 protein Homo sapiens cDNA: FLJ23224fis, clone ADSU02206 dynein, cytoplasmic, intermediate polypeptide 1ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta A (activin A,activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 18-7 Genes that were shown to have increased expression in theICBM- derived cells as compared to the other cell lines assayed. cardiacankyrin repeat protein MHC class I region ORF integrin, alpha 10hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-inducedprotein 44 SRY (sex determining region Y)-box 9 (campomelic dysplasia,autosomal sex-reversal) keratin associated protein 1-1 hippocalcin-like1 jagged 1 (Alagille syndrome) Proteoglycan 1, secretory granule

TABLE 18-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. interleukin 26maltase-glucoamylase (alpha-glucosidase) nuclear receptor subfamily 4,group A, member 2 v-fos FBJ murine osteosarcoma viral oncogene homologhypothetical protein DC42 nuclear receptor subfamily 4, group A, member2 FBJ murine osteosarcoma viral oncogene homolog B WNT1 induciblesignaling pathway protein 1 MCF.2 cell line derived transformingsequence potassium channel, subfamily K, member 15 cartilagepaired-class homeoprotein 1 Homo sapiens cDNA FLJ12232 fis, cloneMAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone LIVER2000775 jun Bproto-oncogene B-cell CLL/lymphoma 6 (zinc finger protein 51) zincfinger protein 36, C3H type, homolog (mouse)

The present example was performed to provide a molecularcharacterization of the cells derived from umbilical cord and placenta.This analysis included cells derived from three different umbilicalcords and three different placentas. The study also included twodifferent lines of dermal fibroblasts, three lines of mesenchymal stemcells, and three lines of iliac crest bone marrow cells. The mRNA thatwas expressed by these cells was analyzed on a GENECHIP oligonucleotidearray that contained oligonucleotide probes for 22,000 genes.

The analysis revealed that transcripts for 290 genes were present indifferent amounts in these five different cell types. These genesinclude seven genes specifically increased in the umbilicaltissue-derived cells and ten genes that are specifically increased inthe placenta-derived cells. Fifty-four genes were found to havespecifically lower expression levels in placenta-derived and umbilicalcord-derived cells.

The expression of selected genes has been confirmed by PCR, as shown inExample 18. Postpartum-derived cells generally, and umbilical derivedcells, in particular, have distinct gene expression profiles, forexample, as compared to other human cells, such as the bonemarrow-derived cells and fibroblasts tested here.

EXAMPLE 19 Cell Markers in Umbilical Cord Tissue-Derived Cells

Gene expression profiles of cells derived from umbilical cord werecompared with those of cells derived from other sources using anAffymetrix GENECHIP. “Signature” genes were identified: interleukin-8(IL-8), reticulon, chemokine receptor ligand 3 (CXC ligand 3), chemokinereceptor (CXC motif) ligand 1, tumor necrosis factor, alpha-inducedprotein 3 and granulocyte chemotactic protein 2 (GCP-2). These“signature” genes were expressed at relatively high levels inumbilicus-derived cells.

The procedures described in this example were conducted to verify themicroarray data and compare data for gene and protein expression, aswell as to establish a series of reliable assays for detection of uniqueidentifiers for umbilical cord tissue-derived cells.

Umbilicus-derived cells (four isolates), and normal human dermalfibroblasts (NHDF; neonatal and adult) were grown in growth medium ingelatin-coated T75 flasks. mesenchymal stem cells (MSCs) were grown inmesenchymal stem cell growth medium Bullet kit (MSCGM; Cambrex,Walkerville, Md.).

For IL-8 experiments, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours ingrowth medium and then grown further for 8 hours in 10 milliliters ofserum starvation medium [DMEM—low glucose (Gibco, Carlsbad, Calif.),penicillin (50 Units/milliliter), streptomycin (50micrograms/milliliter)(Gibco) and 0.1% (w/v) Bovine Serum Albumin (BSA;Sigma, St. Louis, Mo.)]. RNA was then extracted and the supernatantswere centrifuged at 150×g for 5 minutes to remove cellular debris.Supernatants were frozen at −80° C. until ELISA analysis.

The umbilical cord tissue-derived cells, as well as human fibroblastsderived from human neonatal foreskin, were cultured in growth medium ingelatin-coated T75 flasks. Cells were frozen at passage 11 in liquidnitrogen. Cells were thawed and transferred to 15 milliliter centrifugetubes. After centrifugation at 150×g for 5 minutes, the supernatant wasdiscarded. Cells were resuspended in 4 milliliters culture medium andcounted. Cells were grown in a 75 cm² flask containing 15 milliliters ofgrowth medium at 375,000 cell/flask for 24 hours. The medium was changedto a serum starvation medium for 8 hours. Serum starvation medium wascollected at the end of incubation, centrifuged at 14,000×g for 5minutes (and stored at −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftrypsin/EDTA (Gibco, Carlsbad, Calif.) were added to each flask. Aftercells detached from the flask, trypsin activity was neutralized with 8milliliters of growth medium. Cells were transferred to a 15 millilitercentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter growth medium was added to each tube toresuspend the cells. Cell number was determined with a hemocytometer.

The amount of IL-8 secreted by the cells into serum starvation mediumwas analyzed using ELISA assays (R&D Systems, Minneapolis, Minn.). Allassays were conducted according to the instructions provided by themanufacturer.

RNA was extracted from confluent umbilical cord-derived cells andfibroblasts, or for IL-8 expression, from cells treated as describedabove. Cells were lysed with 350 microliters buffer RLT containingbeta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 Units/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50microliters DEPC-treated water and stored at −80° C. RNA was alsoextracted from human umbilical cord. Tissue (30 milligrams) wassuspended in 700 microliters of buffer RLT containingbeta-mercaptoethanol. Samples were mechanically homogenized and the RNAextraction proceeded according to manufacturer's specification. RNA wasextracted with 50 microliters of DEPC-treated water and stored at −80°C.

RNA was reverse-transcribed using random hexamers with the TaqManreverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95° C. for 10minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in umbilicalcord cells (signature genes—including oxidized LDL receptor,interleukin-8, renin, and reticulon), were further investigated usingreal-time and conventional PCR.

PCR was performed on cDNA samples using gene expression products soldunder the tradename Assays-On-Demand (Applied Biosystems) geneexpression products. Oxidized LDL receptor (Hs00234028); renin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH were mixed with cDNA andTaqMan Universal PCR master mix according to the manufacturer'sinstructions (Applied Biosystems) using a 7000 sequence detection systemwith ABI Prism 7000 SDS software (Applied Biosystems). Thermal cycleconditions were initially 50° C. for 2 minutes and 95° C. for 10minutes, followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1minute. PCR data were analyzed according to manufacturer'sspecifications (User Bulletin #2 from Applied Biosystems for ABI Prism7700 Sequence Detection System).

Conventional PCR was performed using an ABI PRISM 7700 (Perkin ElmerApplied Biosystems, Boston, Mass.) to confirm the results from real-timePCR. PCR was performed using 2 microliters of cDNA solution (1×Taqpolymerase (tradename AMPLITAQ GOLD) universal mix PCR reaction buffer(Applied Biosystems) and initial denaturation at 94° C. for 5 minutes.Amplification was optimized for each primer set. For IL-8, CXC ligand 3,and reticulon (94° C. for 15 seconds, 55° C. for 15 seconds and 72° C.for 30 seconds for 30 cycles); for renin (94° C. for 15 seconds, 53° C.for 15 seconds and 72° C. for 30 seconds for 38 cycles); for oxidizedLDL receptor and GAPDH (94° C. for 15 seconds, 55° C. for 15 seconds and72° C. for 30 seconds for 33 cycles). Primers used for amplification arelisted in Table 19-1. Primer concentration in the final PCR reaction was1 micromolar except for GAPDH which was 0.5 micromolar. GAPDH primerswere the same as for real-time PCR, except that the manufacturer'sTaqMan probe was not added to the final PCR reaction. Samples wereseparated on 2% (w/v) agarose gel and stained with ethidium bromide(Sigma, St. Louis, Mo.). Images were captured on 667 film (UniversalTwinpack, VWR International, South Plainfield, N.J.) using a fixedfocal-length POLAROID camera (VWR International, South Plainfield,N.J.).

TABLE 19-1 Primers used Gene of Interest Primers Oxidized LDL S:5′-GAGAAATCCAAAGAGCAAATGG-3′ receptor (SEQ ID NO: 1) A:5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) Renin S:5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′(SEQ ID NO: 4) Reticulon S: 5′- TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5)A: 5′- AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO: 6) Interleukin-8 S:5′- TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7) A:5′-CTTCAAAAACTTCTCCACAACC- 3′ (SEQ ID NO: 8) Chemokine (CXC) S:5′- CCCACGCCACGCTCTCC-3′ ligand 3 (SEQ ID NO: 9) A:5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO: 10) S = Sense, A = Anti-sense

Umbilical cord-derived cells were fixed with cold 4% (w/v)paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at roomtemperature. One isolate each of umbilical cord-derived cells at passage0 (P0) (directly after isolation) and passage 11 (P11) (two isolates ofUmbilical cord-derived cells) and fibroblasts (P11) were used.Immunocytochemistry was performed using antibodies directed against thefollowing epitopes: vimentin (1:500, Sigma, St. Louis, Mo.), desmin(1:150; Sigma—raised against rabbit; or 1:300; Chemicon, Temecula,Ca.—raised against mouse), alpha-smooth muscle actin (SMA; 1:400;Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF;1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested onpassage 11 umbilical cord-derived cells: anti-human GROalpha—PE (1:100;Becton Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; SantaCruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1(ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A (1:100;Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure toprevent epitope loss. Furthermore, in instances where the primaryantibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v)donkey serum was used in place of goat serum throughout the process.Primary antibodies, diluted in blocking solution, were then applied tothe cultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution (no 1° control). Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Adherent cells in flasks were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspended3% (v/v) FBS in PBS at a cell concentration of 1×10⁷/milliliter. Onehundred microliter aliquots were delivered to conical tubes. Cellsstained for intracellular antigens were permeabilized with Perm/Washbuffer (BD Pharmingen, San Diego, Calif.). Antibody was added toaliquots as per manufacturer's specifications, and the cells wereincubated for in the dark for 30 minutes at 4° C. After incubation,cells were washed with PBS and centrifuged to remove excess antibody.Cells requiring a secondary antibody were resuspended in 100 microliterof 3% FBS. Secondary antibody was added as per manufacturer'sspecification, and the cells were incubated in the dark for 30 minutesat 4° C. After incubation, cells were washed with PBS and centrifuged toremove excess secondary antibody. Washed cells were resuspended in 0.5milliliter PBS and analyzed by flow cytometry. The following antibodieswere used: oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa(555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 andM-5284; Sigma), and Donkey against Goat IgG (sc-3743; Santa Cruz,Biotech.). Flow cytometry analysis was performed with FACScalibur(Becton Dickinson San Jose, Calif.).

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human umbilical cord, adult and neonatalfibroblasts, and Mesenchymal Stem Cells (MSCs) indicate that bothreticulon and oxidized LDL receptor expression were higher inumbilicus-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the ΔΔCT method and expressed on alogarithmic scale. No significant differences in the expression levelsof CXC ligand 3 and GCP-2 were found between postpartum cells andcontrols. The results of real-time PCR were confirmed by conventionalPCR. Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum cells and controls using conventional PCR CXC ligand3 primers listed in Table 19-1.

The expression of the cytokine, IL-8 in umbilical cord tissue-derivedcells was elevated in both growth medium-cultured and serum-starvedumbilical cord-derived cells. All real-time PCR data were validated withconventional PCR and by sequencing PCR products.

After growth in serum-free media, the conditioned media were examinedfor the presence of IL-8. The greatest amounts of IL-8 were detected inmedia in which umbilical cells had been grown (Table 19-2). No IL-8 wasdetected in medium in which human dermal fibroblasts had been grown.

TABLE 19-2 IL-8 protein(pg/10⁶ cells) measured by ELISA Cell type IL-8Human fibroblasts ND Placenta Isolate 1 ND UMBC Isolate 1 2058.42 +144.67  Placenta Isolate 2 ND UMBC Isolate 2 2368.86 + 22.73  PlacentaIsolate3 (normal 17.27 + 8.63  O₂) Placenta Isolate 3 (low O₂, 264.92 +9.88  W/O BME) Results of the ELISA assay for interleukin-8 (IL-8)performed on placenta- and umbilical cord-derived cells as well as humanskin fibroblasts. Values are presented here are picogram/million cells,n = 2, sem. ND: Not Detected

Cells derived from the human umbilical cord at passage 0 were probed forthe production of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilical cord-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

The production of GROalpha, GCP-2, oxidized LDL receptor 1 and reticulon(NOGO-A) in umbilical cord-derived cells at passage 11 was investigatedby immunocytochemistry. Umbilical cord-derived cells were GCP-2positive, but GRO alpha production was not detected by this method.Furthermore, cells were NOGO-A positive.

Accordance between gene expression levels measured by microarray and PCR(both real-time and conventional) has been established for cells derivedfrom umbilical cord tissue for two genes: reticulon, and IL-8. Theexpression of these genes was differentially regulated at the mRNA levelin umbilical cord-derived cells, with IL-8 also differentially regulatedat the protein level. Differential expression of GCP-2 and CXC ligand 3was not confirmed at the mRNA level. Although this result does notsupport data originally obtained from the microarray experiment, thismay be due to a difference in the sensitivity of the methodologies.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of alpha-smooth muscle actin and vimentin, and werepositive for both. The staining pattern was preserved through passage11.

In conclusion, the complete mRNA data at least partially verifies thedata obtained from the microarray experiments.

EXAMPLE 20 Immunohistochemical Characterization of Cellular Phenotypes

The phenotypes of cells found within human umbilical cord were analyzedby immunohistochemistry.

Human umbilical cord tissue was harvested and immersion fixed in 4%(w/v) paraformaldehyde overnight at 4° C. Immunohistochemistry wasperformed using antibodies directed against the following epitopes (See,Table 20-1): vimentin (1:500; Sigma, St. Louis, Mo.), desmin (1:150,raised against rabbit; Sigma; or 1:300, raised against mouse; Chemicon,Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400; Sigma),cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200;Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested:anti-human GROalpha-PE (1:100; Becton Dickinson, Franklin Lakes, N.J.),anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.),anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa CruzBiotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 microns thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

Immunohistochemistry was performed similar to previous studies (e.g.,Messina, et al., Exper. Neurol., 2003; 184:816-829). Tissue sectionswere washed with phosphate-buffered saline (PBS) and exposed to aprotein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon,Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1hour to access intracellular antigens. In instances where the epitope ofinterest would be located on the cell surface (CD34, ox-LDL R1), tritonwas omitted in all steps of the procedure to prevent epitope loss.Furthermore, in instances where the primary antibody was raised againstgoat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was used in placeof goat serum throughout the procedure. Primary antibodies, diluted inblocking solution, were then applied to the sections for a period of 4hours at room temperature. Primary antibody solutions were removed, andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing block along with goatanti-mouse IgG-Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/orgoat anti-rabbit IgG-Alexa 488 (1:250; Molecular Probes) or donkeyanti-goat IgG-FITC (1:150; Santa Cruz Biotech). Cultures were washed,and 10 micromolar DAPI (Molecular Probes) was applied for 10 minutes tovisualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epifluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

TABLE 20-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Vimentin 1:500 Sigma, St. Louis, Mo. Desmin (rb) 1:150 SigmaDesmin (m) 1:300 Chemicon, Temecula, Ca. alpha-smooth muscle actin 1:400Sigma (SMA) Cytokeratin 18 (CK18) 1:400 Sigma von Willebrand factor1:200 Sigma (vWF) CD34 III 1:100 DakoCytomation, Carpinteria, Ca.GROalpha-PE 1:100 BD, Franklin Lakes, N.J. GCP-2 1:100 Santa CruzBiotech Ox-LDL R1 1:100 Santa Cruz Biotech NOGO-A 1:100 Santa CruzBiotech

Vimentin, desmin, SMA, CK18, vWF, and CD34 markers were expressed in asubset of the cells found within umbilical cord (data not shown). Inparticular, vWF and CD34 expression were restricted to blood vesselscontained within the cord. CD34+ cells were on the innermost layer(lumen side). Vimentin expression was found throughout the matrix andblood vessels of the cord. SMA was limited to the matrix and outer wallsof the artery and vein, but not contained within the vessels themselves.CK18 and desmin were observed within the vessels only, desmin beingrestricted to the middle and outer layers.

Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, vonWillebrand Factor, and CD34 are expressed in cells within humanumbilical cord. Based on in vitro characterization studies showing thatonly vimentin and alpha-smooth muscle actin are expressed, the datasuggests that the current process of umbilical cord-derived cellisolation harvests a subpopulation of cells or that the cells isolatedchange expression of markers to express vimentin and alpha-smooth muscleactin.

EXAMPLE 21 Secretion of Trophic Factors

The secretion of selected trophic factors from umbilicus-derived cellswas measured. Factors selected for detection included: (1) those knownto have angiogenic activity, such as hepatocyte growth factor (HGF)(Rosen et al., Ciba Found. Symp., 1997; 212:215-26), monocytechemotactic protein 1 (MCP-1) (Salcedo et al., Blood, 2000; 96:34-40),interleukin-8 (IL-8) (Li et al., J. Immunol., 2003; 170:3369-76),keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF) (Hughes et al., Ann. Thorac.Surg., 2004; 77:812-8), matrix metalloproteinase 1 (TIMP1), angiopoietin2 (ANG2), platelet derived growth factor (PDGF-bb), thrombopoietin(TPO), heparin-binding epidermal growth factor (HB-EGF), stromal-derivedfactor 1alpha (SDF-1alpha); (2) those known to haveneurotrophic/neuroprotective activity, such as brain-derivedneurotrophic factor (BDNF) (Cheng et al., Dev. Biol., 2003; 258;319-33), interleukin-6 (IL-6), granulocyte chemotactic protein-2(GCP-2), transforming growth factor beta2 (TGFbeta2); and (3) thoseknown to have chemokine activity, such as macrophage inflammatoryprotein 1alpha (MIP1a), macrophage inflammatory protein 1beta (MIP1b),monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation,normal T cell expressed and secreted), I309, thymus andactivation-regulated chemokine (TARC), Eotaxin, macrophage-derivedchemokine (MDC), and IL-8.

Cells derived from umbilical cord, as well as human fibroblasts derivedfrom human neonatal foreskin, were cultured in growth medium ongelatin-coated T75 flasks. Cells were cryopreserved at passage 11 andstored in liquid nitrogen. After thawing, growth medium was added to thecells, followed by transfer to a 15 milliliter centrifuge tube andcentrifugation of the cells at 150×g for 5 minutes. The cell pellet wasresuspended in 4 milliliters growth medium, and cells were counted.Cells were seeded at 5,000 cells/cm² in T75 flasks each containing 15milliliters of growth medium, and cultured for 24 hours. The medium waschanged to a serum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v)bovine serum albumin (Sigma), penicillin (50 Units/milliliter) andstreptomycin (50 micrograms/milliliter, Gibco)) for 8 hours. Conditionedserum-free medium was collected at the end of incubation bycentrifugation at 14,000×g for 5 minutes and stored at −20° C.

To estimate the number of cells in each flask, cells were washed withphosphate-buffered saline (PBS) and detached using 2 milliliterstrypsin/EDTA (Gibco). Trypsin activity was inhibited by addition of 8milliliters growth medium. Cells were centrifuged at 150×g for 5minutes. The supernatant was removed, and cells were resuspended in 1milliliter growth medium. Cell number was estimated with ahemocytometer.

Cells were grown at 37° C. in 5% carbon dioxide and atmospheric oxygen.The amount of MCP-1, IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta2produced by each cell sample was determined by ELISA (R&D Systems,Minneapolis, Minn.). All assays were performed according to themanufacturer's instructions. Values presented are picograms permilliliter per million cells (n=2, sem).

Chemokines (MIP1alpha, MIP1beta, MCP-1, Rantes, I309, TARC, Eotaxin,MDC, IL8), BDNF, and angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1,ANG2, PDGFbb, TPO, HB-EGF were measured using SEARCHLIGHT ProteomeArrays (Pierce Biotechnology Inc.). The Proteome Arrays are multiplexedsandwich ELISAs for the quantitative measurement of two to sixteenproteins per well. The arrays are produced by spotting a 2×2, 3×3, or4×4 pattern of four to sixteen different capture antibodies into eachwell of a 96-well plate. Following a sandwich ELISA procedure, theentire plate is imaged to capture the chemiluminescent signal generatedat each spot within each well of the plate. The signal generated at eachspot is proportional to the amount of target protein in the originalstandard or sample.

MCP-1 and IL-6 were secreted by umbilicus-derived cells and dermalfibroblasts (Table 21-1). SDF-1alpha and GCP-2 were secreted byfibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived cells.TGF-beta2 was not detected from either cell type by ELISA.

TABLE 21-1 ELISA assay results (values presented are picog/ml/millioncells (n = 2, sem) MCP-1 IL-6 VEGF SDF-1α GCP-2 IL-8 TGF-beta2Fibroblast  17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Umbilicus (022803)1150 ± 74 4234 ± 289 ND ND 160 ± 11 2058 ± 145 ND Umbilicus (071003)2794 ± 84 1356 ± 43  ND ND 2184 ± 98  2369 ± 23  ND Key: ND: NotDetected.

TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1, RANTES, I309, TARC,MDC, and IL-8 were secreted from umbilicus-derived cells (Tables 21-2and 21-3). No Ang2, VEGF, or PDGF-bb were detected.

TABLE 21-2 SEARCHLIGHT Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF Hfb 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7 ND 9.3 165.7 U3 21850.0ND ND 1134.5 9.0 195.6 30.8 ND 5.4 388.6 Key: hFB (human fibroblasts),U1 (umbilicus-derived cells (022803)), U3 (umbilicus-derived cells(071003)). ND: Not Detected.

TABLE 21-3 SEARCHLIGHT Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 U1ND 8.0 1694.2 ND 22.4 37.6 ND 18.9 51930.1 U3 ND 5.2 2018.7 41.5 11.621.4 ND 4.8 10515.9 Key: hFB (human fibroblasts), U1 (umbilicus-derivedPPDC (022803)), U3 (umbilicus-derived PPDC (071003)). ND: Not Detected.

Umbilicus-derived cells secreted a number of trophic factors. Some ofthese trophic factors, such as HGF, bFGF, MCP-1 and IL-8, play importantroles in angiogenesis. Other trophic factors, such as BDNF and IL-6,have important roles in neural regeneration.

EXAMPLE 22 In Vitro Immunology

Umbilical cord cell lines were evaluated in vitro for theirimmunological characteristics in an effort to predict the immunologicalresponse, if any, these cells would elicit upon in vivo transplantation.Umbilical cord cell lines were assayed by flow cytometry for theexpression of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. Theseproteins are expressed by antigen-presenting cells (APC) and arerequired for the direct stimulation of naïve CD4⁺ T cells (Abbas &Lichtman, Cellular and Molecular Immunology, 5th Ed. (2003) Saunders,Philadelphia, p. 171). The cell lines were also analyzed by flowcytometry for the expression of HLA-G (Abbas & Lichtman, Cellular andMolecular Immunology, 5th Ed. (2003) Saunders, Philadelphia, p. 171),CD178 (Coumans, et. al., Journal of Immunological Methods, 1999;224:185-196), and PD-L2 (Abbas & Lichtman, Cellular and MolecularImmunology, 5th Ed. (2003) Saunders, Philadelphia, p. 171; Brown, et.al., The Journal of Immunology, 2003; 170:1257-1266). The expression ofthese proteins by cells residing in placental tissues is thought tomediate the immuno-privileged status of placental tissues in utero. Topredict the extent to which umbilical cord tissue-derived cell lineselicit an immune response in vivo, the cell lines were tested in aone-way mixed lymphocyte reaction (MLR).

Cells were cultured in growth medium (DMEM-low glucose (Gibco, Carlsbad,Calif.), 15% (v/v) fetal bovine serum (FBS); (Hyclone, Logan, Utah.),0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco, Carlsbad, Calif.) until confluent in T75 flasks (Corning,Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cellswere harvested, centrifuged, and re-suspended in 3% (v/v) FBS in PBS ata cell concentration of 1×10⁷ per milliliter. Antibody (Table 22-1) wasadded to one hundred microliters of cell suspension as permanufacturer's specifications and incubated in the dark for 30 minutesat 4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were re-suspended in five hundredmicroliters of PBS and analyzed by flow cytometry using a FACS caliburinstrument (Becton Dickinson, San Jose, Calif.).

TABLE 22-1 Antibodies Antibody Manufacturer Catalog Number HLA-DRDPDQ BDPharmingen (San Diego, Ca.) 555558 CD80 BD Pharmingen (San Diego, Ca.)557227 CD86 BD Pharmingen (San Diego, Ca.) 555665 B7-H2 BD Pharmingen(San Diego, Ca.) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100CD178 Santa Cruz (San Cruz, Ca.) sc-19681 PD-L2 BD Pharmingen (SanDiego, Ca.) 557846 Mouse IgG2a Sigma (St. Louis, Mo) F-6522 MouseIgG1kappa Sigma (St. Louis, Mo.) P-4685

Cryopreserved vials of passage 10 umbilical cord-derived cells labeledas cell line A were packaged on dry ice and sent to CTBR (Senneville,Quebec) to conduct a mixed lymphocyte reaction using CTBR SOP no.CAC-031. Peripheral blood mononuclear cells (PBMCs) were collected frommultiple male and female volunteer donors. Stimulator (donor) allogeneicPBMC, autologous PBMC, and umbilical cord tissue-derived cell lines weretreated with mitomycin C. Autologous and mitomycin C-treated stimulatorcells were added to responder (recipient) PBMCs and cultured for 4 days.After incubation, [³H]thymidine was added to each sample and culturedfor 18 hours. Following harvest of the cells, radiolabeled DNA wasextracted, and [³H]-thymidine incorporation was measured using ascintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the umbilical cord-derived cells was calculatedas the mean proliferation of the receiver plus mitomycin C-treatedumbilical cord tissue-derived cell line divided by the baselineproliferation of the receiver.

Six human volunteer blood donors were screened to identify a singleallogeneic donor that will exhibit a robust proliferation response in amixed lymphocyte reaction with the other five blood donors. This donorwas selected as the allogeneic positive control donor. The remainingfive blood donors were selected as recipients. The allogeneic positivecontrol donor and umbilical cord-derived cell lines were mitomycinC-treated and cultured in a mixed lymphocyte reaction with the fiveindividual allogeneic receivers. Reactions were performed in triplicateusing two cell culture plates with three receivers per plate (Table22-2). The average stimulation index ranged from 6.5 (plate 1) to 9(plate 2) and the allogeneic donor positive controls ranged from 42.75(plate 1) to 70 (plate 2) (Table 22-3).

TABLE 22-2 Mixed Lymphocyte Reaction Data-Cell Line A (Umbilical Cord)DPM for Proliferation Assay Analytical Culture Replicates number System1 2 3 Mean SD CV Plate ID: Plate 1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic Control of autostimulation (Mitomycin treated autologouscells) 567 604 374 515.0 123.50 24.0 donor) Cell line Proliferationbaseline of receiver 5101 3735 2973 3936.3 1078.19 27.4 type A Controlof autostimulation (Mitomycin treated autologous cells) 1924 4570 21532882.3 1466.04 50.9

TABLE 22-3 Average stimulation index of umbilical cord-derived cells andan allogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Recipient Umbilical Cord Plate 1 (receivers 1-4)42.75 6.5 Plate 2 (receiver 5) 70 9

Histograms of umbilical cord-derived cells analyzed by flow cytometryshow negative expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, asnoted by fluorescence value consistent with the IgG control, indicatingthat umbilical cord-derived cell lines lack the cell surface moleculesrequired to directly stimulate allogeneic PBMCs (e.g., CD4⁺ T cells).

Histograms of umbilical cord-derived cells analyzed by flow cytometryshow positive expression of PD-L2, as noted by the increased value offluorescence relative to the IgG control, and negative expression ofCD178 and HLA-G, as noted by fluorescence value consistent with the IgGcontrol.

In the mixed lymphocyte reactions conducted with umbilical cord-derivedcell lines, the average stimulation index ranged from 6.5 to 9, and thatof the allogeneic positive controls ranged from 42.75 to 70. Umbilicalcord-derived cell lines were negative for the expression of thestimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, asmeasured by flow cytometry. Umbilical cord-derived cell lines werenegative for the expression of immuno-modulating proteins HLA-G andCD178 and positive for the expression of PD-L2, as measured by flowcytometry. Allogeneic donor PBMCs contained antigen-presenting cellsexpressing HLA-DP, DR, DQ, CD80, CD86, and B7-H2, thereby allowing forthe stimulation of allogeneic PBMCs (e.g., naïve CD4⁺ T cells). Theabsence of antigen-presenting cell surface molecules on umbilicalcord-derived cells required for the direct stimulation of allogeneicPBMCs (e.g., naïve CD4⁺ T cells) and the presence of PD-L2, animmuno-modulating protein, may account for the low stimulation indexexhibited by these cells in a MLR as compared to allogeneic controls.

EXAMPLE 23 Assay for Telomerase Activity

Telomerase functions to synthesize telomere repeats that serve toprotect the integrity of chromosomes and to prolong the replicative lifespan of cells (Liu, K, et al., PNAS, 1999; 96:5147-5152). Telomeraseconsists of two components, telomerase RNA template (hTER) andtelomerase reverse transcriptase (hTERT). Regulation of telomerase isdetermined by transcription of hTERT but not hTER. Real-time polymerasechain reaction (PCR) for hTERT mRNA thus is an accepted method fordetermining telomerase activity of cells.

Cell Isolation

Real-time PCR experiments were performed to determine telomeraseproduction of human umbilical cord tissue-derived cells. Human umbilicalcord tissue-derived cells were prepared in accordance with Examples13-15 and the examples set forth in U.S. application Ser. No. 10/877,012(the '012 application), which issued as U.S. Pat. No. 7,510,873.Generally, umbilical cords obtained from National Disease ResearchInterchange (Philadelphia, Pa.) following a normal delivery were washedto remove blood and debris and mechanically dissociated. The tissue wasthen incubated with digestion enzymes including collagenase, dispase andhyaluronidase in culture medium at 37° C. human umbilical cordtissue-derived cells were cultured according to the methods set forth inthe examples of the '012 application. Mesenchymal stem cells and normaldermal skin fibroblasts (cc-2509 lot #9F0844) were obtained fromCambrex, Walkersville, Md. A pluripotent human testicular embryonalcarcinoma (teratoma) cell line nTera-2 cells (NTERA-2 cl.D1), (see,Plaia et al., Stem Cells, 2006; 24(3):531-546) was purchased from ATCC(Manassas, Va.) and was cultured according to the methods set forth inthe '012 application.

Total RNA Isolation

RNA was extracted from the cells using RNeasy® kit (Qiagen, Valencia,Calif.). RNA was eluted with 50 microliters DEPC-treated water andstored at −80° C. RNA was reverse transcribed using random hexamers withthe TaqMan® reverse transcription reagents (Applied Biosystems, FosterCity, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes and 95° C.for 10 minutes. Samples were stored at −20° C.

Real-Time PCR

PCR was performed on cDNA samples using the Applied BiosystemsAssays-On-Demand™ (also known as TaqMan® Gene Expression Assays)according to the manufacturer's specifications (Applied Biosystems).This commercial kit is widely used to assay for telomerase in humancells. Briefly, hTERT (human telomerase gene) (Hs00162669) and humanGAPDH (an internal control) were mixed with cDNA and TaqMan® UniversalPCR master mix using a 7000 sequence detection system with ABI prism7000 SDS software (Applied Biosystems). Thermal cycle conditions wereinitially 50° C. for 2 min and 95° C. for 10 min followed by 40 cyclesof 95° C. for 15 sec and 60° C. for 1 min. PCR data was analyzedaccording to the manufacturer's specifications.

Human umbilical cord tissue-derived cells (ATCC Accession No. PTA-6067),fibroblasts, and mesenchymal stem cells were assayed for hTERT and 18SRNA. As shown in Table 22-1, hTERT, and hence telomerase, was notdetected in human umbilical cord tissue-derived cells.

TABLE 22-1 hTERT 18S RNA Umbilical cells (022803) ND + Fibroblasts ND +ND-not detected; + signal detected

Human umbilical cord tissue-derived cells (isolate 022803, ATCCAccession No. PTA-6067) and nTera-2 cells were assayed and the resultsshowed no expression of the telomerase in two lots of hUTC while theteratoma cell line revealed high level of expression (Table 22-1).

TABLE 22-1 hTERT GAPDH Cell type Exp.1 Exp.2 Exp.1 Exp.2 hTERT normnTera2 22.85 27.31 16.41 16.31 .61 022803 — — 22.97 22.79 —

Therefore, it can be concluded that human umbilical tissue-derived cellsdo not express telomerase.

What is claimed:
 1. A method of treating a patient having a neurological injury comprising administering to the patient isolated umbilical cord tissue-derived cells in an amount effective to treat the neurological injury, wherein the neurological injury is cerebral hemorrhage, cerebral ischemia or traumatic brain injury, and wherein the umbilical cord tissue-derived cells are derived from human umbilical cord tissue substantially free of blood, are capable of self-renewal and expansion in culture, have the potential to differentiate into cells of at least a neural phenotype, and do not express CD117.
 2. The method of claim 1, wherein the cells are administered with at least one other cell type.
 3. The method of claim 2, wherein the other cell type is an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell or other multipotent or pluripotent stem cell.
 4. The method of claim 1, wherein the umbilical cord tissue-derived cells are administered at a pre-determined site in the central nervous system of the patient.
 5. The method of claim 1, wherein the umbilical cord tissue-derived cells are administered by injection or infusion.
 6. The method of claim 1, wherein the umbilical cord tissue-derived cells further express CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C.
 7. The method of claim 1, wherein the umbilical cord tissue-derived cells further do not express CD31, CD34, CD45, CD141 and HLA-DR, DP, DQ.
 8. The method of claim 1, wherein the neurological injury is cerebral hemorrhage.
 9. The method of claim 1, wherein the neurological injury is cerebral ischemia.
 10. The method of claim 1, wherein the neurological injury is traumatic brain injury.
 11. A method of stimulating regeneration capacity of a subventricular zone of a patient following traumatic brain injury comprising administering to the patient isolated umbilical cord tissue-derived cells in an amount effective to increase neurogenesis, angiogenesis, or synaptogenesis, wherein the umbilical cord tissue-derived cells are derived from human umbilical cord tissue substantially free of blood, are capable of self-renewal and expansion in culture, have the potential to differentiate into cells of at least a neural phenotype, and do not express CD117.
 12. The method of claim 11, wherein the umbilical cord tissue-derived cells are administered with at least one other cell type.
 13. The method of claim 12, wherein the other cell type is an astrocyte, oligodendrocyte, neuron, neural progenitor, neural stem cell or other multipotent or pluripotent stem cell.
 14. The method of claim 11, wherein the cells are administered at a pre-determined site in the central nervous system of the patient.
 15. The method of claim 11, wherein the cells are administered by injection or infusion.
 16. The method of claim 11, wherein the umbilical cord tissue-derived cells further express CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C.
 17. The method of claim 11, wherein the umbilical cord tissue-derived cells further do not express CD31, CD34, CD45, CD141 and HLA-DR, DP, DQ. 